The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-Å resolution

PROTEINS Structure, Function, and Genetics 2548-78 (1996)

The Three-Dimensional Structure of Escherichia coli Porphobilinogen Deaminase at 1.76-A Resolution Gordon V. Louie,' Paul D. Brownlie,' Richard Lambert,' Jonathan B. Cooper,' Tom L. Blundell,' Steve P. Wood,' Vladimir N. Malashkevich? Alfons Hadener? Martin J. Warren: and Peter M. Shoolingin-Jordan4 'Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, London WClE 7HX, United Kingdom; 'Znstitut fur Organische Chemie der Uniuersitat, CH4056 Basel, Switzerland; 3Department of Molecular Genetics, Institute of Ophthalmology, London ECl V 9EL, United Kingdom; 4Department of Biochemistry, University of Southampton, Southampton SO1 6 7PX, United Kingdom

ABSTRACT Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the l-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild- type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 A resolution. The polypeptide chain of PBGD is folded into three d P domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed P-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with sele- nomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the

0 1996 WILEY-LISS, INC.

positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommo- dation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains l and 2 to carry the terminal ring into the appropriate position at the catalytic site, or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three- dimensional structure and mechanism of activity. o 1996 Wiley-Liss, h c .

Key words: prophobilinogen deaminase, E. coli, selenomethionine, X-ray analysis, tetrapyrrole biosynthesis

INTRODUCTION The enzyme porphobilinogen deaminase (PBGD;

EC 4.3.1.8, known also as 1-hydroxymethylbilane synthase or uroporphyrinogen I synthase) is an early enzyme in the tetrapyrrole biosynthetic pathway. PBGD catalyzes the step-wise, head-to-tail polymerization of four molecules of porphobilinogen to yield the 1-hydroxymethylbilane, preuroporphyrinogen (see Fig. 1, see reference 1 for review). Uro- porphyrinogen I11 synthase, the next enzyme of the pathway, catalyzes the rearrangement of the terminal pyrrole ring of preuroporphyrinogen and ring-

Received November 16, 1993; revision accepted October 23, 1995.

Address reprint requests to Prof. T.L. Blundell, Laboratory of Molecular Biology, Department of Crystallography, Birk- beck College, University of London, Malet Street, London WClE 7HX. UK.

STRUCTURE OF E . COLI PBGD

P A

- I - - -

P

b L s 2 4 2

49

\ (hydroxymethylbllaneI A

I P

Fig. 1. The tetrapolymerization reaction catalyzed by PBGD, in which four molecules of the substrate porphobilinogen (PBG) are assembled into the 1 -hydroxymethylbilane product, preuroporphyrinogen. The intermediates ES,, ES,, ES,, and ES, are

generated upon sequential incorporation of PBGD molecules in the order a, b, c, and d. The acetate and propionate side-groups of the porphobilinogen moieties are denoted by -A and -P, respectively.

closure to generate uroporphyrinogen 111, the cyclic tetrapyrrole common to the biosynthesis of haems, chlorophylls, corrins, and all other related macrocy- cles. Porphobilinogen deaminase is a monomeric enzyme with M,s ranging from 34,000-45,000 depend- ing on the species.' Relatively high amino-acid sequence conservation is found, amounting to a t least 32% in the proteins from bacteria, fungi, plants, and mammals.

In humans, a deficiency in the activity of PBGD is associated with the hereditary disease acute inter-

mittent porphyria, an autosomal dominant disorder affecting 1 in 10,000 persons.' Recently, the precise genetic defects producing this disease have been identified in a number of patients (see reference 3 for review).

The PBGD enzyme contains a novel dipyrromethane cofactor4, that is assembled by the apoenzyme from two molecules of porphobilinogen and attached to the polypeptide chain through a thioether bond to a cysteine side-chain. The cofactor serves as a primer to which the product polypyrrole

50

-0 k G.V. LOUIE ET AL.

A P

0 - & H q

pomhobilinoaen deamination

' ES,

bond formation gvrrolenine

A P A P

A

Fig. 2. The chemistry of a porphobilinogen ring-coupling reaction. Carbon-carbon bond formation proceeds through (1) deamination of the substrate porphobilinogen (PBG) to yield the methylene pyrrolinene; (2) nucleophilic attack by the methylene carbon

atom at the free a-position of the terminal, enzyme-bound ring; and (3) deprotonation at this same carbon atom. The acetate and propionate side-groups of the porphobilinogen moieties are denoted by -A and -P, respectively.

is attached during the tetrapolymerization reaction. The cofactor is not itself turned over and, once assembled, remains a permanent feature of the enzyme that is important in stabilizing the structure of the protein. The apoenzyme has a lowered electro- phoretic mobility under non-denaturing conditions and exhibits an NMR spectrum more typical of that of a denatured enzyme.5

Stepwise assembly of the tetrapyrrole product, in which the pyrrole rings are added in the order a, b, c, and d,6,7 proceeds via four repetitions of the following reactions: (1) deamination of the incoming porphobilinogen substrate; (2) carbon-carbon bond formation through nucleophilic attack by the carbon atom at the free a-position of the terminal enzyme-bound pyrrole ring; (3) deprotonation at this same carbon atom (see Fig. 2). The initial assembly of the cofactor may occur similarly, except that the attachment of the first porphobilinogen unit (Cl) to the polypeptide chain involves the nucleophilic attack by the -S- of cysteine-242. The C2 ring is then added to complete the dipyrromethane cofactor. As- sembly of the cofactor is accompanied by a conformational change in the protein that results in its permanent and irreversible installation at the active site.

The catalytic reaction proceeds through four covalent enzyme-intermediate complexes, ES, ES,, ES,, and ES,, that are sequentially generated during the course of the tetrapolymerization. These are stable

to various degrees (ES, is the most stable) and are isolable.' The product preuroporphyrinogen is re- leased from ES, by reaction with water essentially through a reversal of the initial ring-coupling reaction: (1) protonation of the carbon at the a-position of the cofactor ring C2; (2) cleavage of the carbon- carbon bond between the cofactor ring C2 and ring a; (3) finally, hydration (rather than amination) of the tetrapyrrolic methylene pyrrolenine to yield preuroporphyrinogen and to regenerate the enzyme (El.

Several other features of the porphobilinogen tetrapolymerization reaction are noteworthy. First, PBGD catalyzes all reaction steps stereospecifi- ~ a l l y . ~ ~ l ~ Second, each of the substrate ring couplings is reversible. Thus the terminal ring of the pyrrole chain (with the important exception of ring C2 of the cofactor) in each of the ES complexes is susceptible to hydrolytic release as hydroxyporphobilinogen. Third, only the monopyrrolic porphobilinogen, and not di- or tripyrroles, can be efficiently used as substrate. Furthermore, synthetic analogues with modified acetate or propionate side-substituents are inactive as substrates, acting instead as competitive inhibitors." Hydroxyporphobilino- gen can be utilized as a substrate but is incorporated at one third of the rate of porphobilinogen itself." Fourth, the exocyclic, carbon-carbon double bond in the tetrapyrrolic methylene pyrrolenine can be in- tercepted at the active site by nucleophiles such as

STRUCTURE OF E. COLZ PBGD 51 TABLE I. Statistics of the X-Ray Data for Native and Heavy Atom Derivative Crystals of PBGD*

~

Reflections R-factor ReS. Phasing Crystal Measured Unique Merg Deriv MaxA Compl power Native 181390 34147 0.010 N/A 1.76 99.0 SeMet 49480 19715 0.080 N/A 2.1 93.8 K,RCl, 30749 5836 0.097 0.243 3.0 85.1 1.06 K,UO,F, 83763 5947 0.166 0.294 3.0 91.7 1.83 UOz(CH,COOH), 31078 5797 0.122 0.345 3.0 88.0 1.86 UO,SO, 23600 5898 0.045 0.197 3.3 89.9 0.81 PCMBS 33237 6018 0.084 0.127 3.3 89.9 0.81 Ybc1, 60844 8149 0.085 0.147 3.3 97.9 0.84 *The phasing analysis used isomorphous and anomalous differences only for the K,PtCl,, K,UO,F,, and UO,(CH,COOH), derivatives. PCMBS, para-chloromercuribenzene sulphonate; Merg, merging; Deriv, derivative, Res. max A, maximum resolution A; Compl, percentage of completeness. Definitions: Merging R-factor =Zh,, Zy= ~~I(hkl)i-I(hkl)~/ Zhkl 1 I(hkl)i Derivative R-factm IF,,,i"(hkl)-F,,(hkl)1/1/2 [F,,,(hkl) + F,,(hkl)l Overall phasing power = [Z,,, Fh2(hkl)/Zh, e2(hk1)1"2

ammonia, hydroxylamine, or methoxyamine, in addition to water. The presence of these bases can cause the premature release of mono-, di-, and tripyrroles from the ES, and ES, enzyme-intermediate c~mplexes . ' ~*~~ Finally, during the course of tetrapolymerization, a bulky and highly acidic, hexapyrrolic intermediate is generated. Site-directed mutagenesis has been exploited in probing the role of arginines in binding the carboxylate groups of the acetate and propionate side-substituents of the polypyrrole chain.15*16

We have reported the crystallization of PBGD from E. colil7 and have given a preliminary description of the three-dimensional structure of this enzyme at 1.9 A." We now report on the structure of the wild-type protein at 1.76 A resolution and of a variant protein in which the six methionine residues have been replaced with ~e1enomethionine.l~ A detailed analysis of the structural features of the PBGD molecule, particularly with respect to the conformation of the dipyrromethane cofactor in the native and oxidized forms, gives insight into the nature of substrate binding. These analyses provide a basis for further insight into the catalytic mechanism of the enzyme.

EXPERIMENTAL METHODS Crystals

Native PBGD from E. coli was prepared2' and cry~tallizedl~ as described previously. The protein was expressed in an E. coli strain BM3 harboring the plasmid pUC18 into which the hemC gene with its own promoter had been cloned. The cell lysate was subjected to a heat treatment and PBGD was purified through a series of chromatographic steps. Crystals were grown in darkness at room temperature in batches of 1 ml volume of solution typically containing 12mg/ml protein, 1% (w/v) sodium chlo- ride, 4% (v/v) acetone, 0.1M sodium acetate buffer (pH5.0), and 9% (w/v) polyethylene glycol 6,000. The

crystals were diamond shaped tiles and deep yellow in color. They belong to the space group P2,2,2 with unit cell dimensions a = 88.0 A, b = 75.9 A, and c = 50.5 A. The c axis lies normal to the plate-face most prominent in the external morphology. There is a single molecule of PBGD per asymmetric unit (M, = 34,270; 313 amino-acid residues). The volume per unit molecular weightz1 V, is 2.46 A3/dalton.

A variant form of PBGD, having quantitative substitution of selenomethionine at each of the six methionine residues, was prepared as described by Ha- dener et a1.l' in order to solve the structure using multi-wavelength anomalous dispersion (MAD) methods (W. Hunter, unpublished data). Crystals of this variant protein, grown under relatively high reducing conditions (-15mM dithiothreitol) were isomorphous with those of the wild-type protein. X-Ray Data

Six suitable heavy-atom derivatives (Table I) were identified that produced significant changes in diffraction intensities on precession photographs. X-Ray intensities for the native protein to 3.0-A resolution, for the selenomethionyl variant and for five of the heavy-atom derivatives, were measured on Enraf-Nonius (Delft, the Netherlands) FAST area detectors, controlled by the program MADNES." The data set for the uranyl sulphate derivative was collected on a Siemens Xentronics area detector (Karlsruhe, Germany), and processed with the XEN- GEN program suite.', The high resolution (1.76 A) data set for the native enzyme was collected with a Arndt-Wonacott camera (Enraf-Nonius) a t the Syn- chrotron Radiation Source (Station 9.6, A = 0.9 A) at Daresbury, U.K. Reflections were indexed and inte- grated with the MOSFLM program suite.

All X-ray intensity data, with the exception of those with uranyl sulphate, were processed with the CCP4 (SERC Daresbury) program suite.24 The scale and temperature factors relating the individual ro-

52 G.V. LOUIE ET AL.

TABLE 11. Heavy Atom Models Used in the Phasing Analysis of PBGD* ~ ~ ~ ~~~~~~

Binding site and Fractional coordinates Derivative (no. binding sites) X Y z Occupancy interactions K2PtC14 (1) 0.2540 0.0423 0.4111 0.91 Pt: near Arg176 K,UO$', (4) 0.2873 0.1713 0.8368 0.97 Ul:Glu163, Glu256

0.4951 0.2383 0.6199 0.55 U2:GLU72, Glu292 0.1859 0.1867 0.8907 0.61 U3:Asp254 0.9862 0.0512 0.4899 0.31 U4:Glu88, Glu239

0.2920 0.1703 0.8446 0.84 u1 0.4857 0.2349 0.6303 0.63 u2

UO~(CH3COOH)~ (3) 0.1875 0.1862 0.8905 0.97 u 3

uozso4 (1) 0.1798 0.1951 0.8887 0.63 u 3

Ybc1, (1) 0.5082 0.2606 0.6105 0.47 u 2

PCMBS (2) 0.2473 0.0577 0.1371 0.22 Hgl:Cys99, His221, Lys26 0.0727 0.063 0.1543 0.26 Hg2:Cys205, Arg213, Val96

*PCMBS, para-chloromercuribenzene sulphonate

tation or oscillation frames were calculated with the least-squares method of Fox and H01mes.'~ The absolute-scale factor and temperature factor for the wild-type and selenomethionyl variant data sets were estimated from a Wilson p l ~ t , ~ ~ , ' ~ as implemented in the program MULTAN.28

Statistics for the X-ray data for all crystals are provided in Table I. For the data on the native protein, the fraction of structure factor amplitudes greater than 3aF drops below 0.6 only at a resolution beyond 1.8 A. For the selenomethionyl variant F z 3crF for 93.4% of the reflections. The R-factor between the native and selenomethionyl variant data, considering the 19,534 reflections in common in the resolution range -2.0 A, was 0.192 on F (0.248 on I). All of the data for the heavy-atom derivatives were placed on the same scale as the native set by a local-scaling pr~cedure.~' For the heavy- atom derivatives, local scaling was also applied to the F+ and F- Friedel-pairs.

Heavy-Atom Models Potential heavy-atom binding sites were identified

from inspection of difference Patterson maps. Auto- mated deconvolution of the Patterson was carried out using symmetry-minimum and vector-superposition analyses, as implemented in the program VECSUM (CCP4 suite). Patterson calculations used pHLE coefficients,3° or AF2 for derivatives with unusable anomalous differences. Additional heavy atom sites were identified in later stages of the analysis from cross-phased difference Fourier maps that were also used to confirm sites initially determined. The absolute configuration of each heavy atom model was assigned based on the relative heights of the enan- tiomerically related peaks in difference Fourier maps cross-phased using the combined isomorphous and anomalous differences from another single deriva- t i ~ e . ~ '

Heavy-atom positions and occupancies were re-

fined initially against the locations and heights of inter-site vectors in the Patterson map (program VECREF, CCP4 suite) and then in reciprocal space against structure-factor amplitudes of centric reflections or against F,,,s (program REFINE). Finally, the heavy-atom models for all six derivatives were refined simultaneously with the program PHARE. Typically, refined positions were found to agree well amongst the refinement procedures, although occupancies varied by as much as 50%. In all cases heavy-atom temperature factors were fixed at 20 A'. The final heavy-atom models used in the phasing analysis are detailed in Table 11. Considerable differences in the relative reactivity of a set of four common sites was evident for the three uranyl com- pounds investigated.

Structure Determination Through Multiple-Isomorphous Replacement

Protein phases and Hendrickson and Lattman31 phase-probability coefficients were calculated (program PHASE, CCP4 suite) from the isomorphous and anomalous differences from four derivatives and the isomorphous differences alone from two derivatives (Table I). The mean figure of merit was 0.73 for 6,315 reflections in the resolution range -3.0 A. Five iterations of solvent flattening:' for which the molecular envelope was determined in reciprocal space33 with an assumed solvent content of 49%, increased the overall mean figure of merit to 0.92. The solvent-flattening procedure produced an overall average shift of 50" from the original MIR phases and an R-factor of 0.208 between F, and F, and improved significantly the interpretability of the map.

The atomic model was fitted against electron-density maps with the program FROD0,34 running on an Evans and Sutherland PS390 (Salt Lake City, UT) or on a Silicon Graphics IRIS4D (Mountain View, CA). Idealized regular polyalanine secondary structural elements were first positioned where in-

STRUCTURE OF E. COLZ PBGD 53 dicated by the electron density, after which connecting loop segments were then fitted. Assignment of amino acid identities" to the backbone (initially 275 of the 313 residues), progressing from two tryp- tophans, was without ambiguity.

Structural Refinement Refinement of the atomic model for wild-type

PBGD proceeded through 14 rounds of standard restrained least-squares [rounds 1-9: RESTRAIN;35 rounds 10-14: PROLSQ.36 One run of simulated annealing was also included [(X-PLOR37 according to the slow-cooling protocol of Brunger and Krukow- ski38] starting from an initial temperature of 2,000"K. X-ray data of increasingly higher resolution were introduced as the refinement progressed. Be- tween rounds of refinement, the atomic model was inspected manually against electron-density maps displayed with FRODO. Maps were calculated with coefficients of the type 2F,-F,, a, and F,-F,, a=. In some cases, values were weighted to compensate for errors due to missing or wrongly positioned atoms in the model, according to the scheme of Read,39 as implemented in the program SIMWT CCP4 suite. Also F,-F,' a,' where F,' and a,' were calculated with a portion of the atomic model omitted. Throughout the course of refinement, reasonably ideal bonding geometry in the atomic model was maintained. No cut-offs on a, have been applied to the reflection data.

The initial MIR model gave an R-factor of 0.438 for 6,102 reflections in the resolution range 10.0-3.0 A. The first round of refinement reduced the R-factor to 0.331. At this point, combination (program SIGMAA CCP4 suite, Read3') of phase information from the atomic model and from the MIRlsolvent-flattening analysis yielded improved electron-density maps that allowed the dipyrromethane cofactor to be built. Refinement of individual atomic temperature factors was initiated in round 3 when 2.5 A resolution data was included. Beginning in round 6, water molecules were added to the atomic model, only at sites where electron density occurred in both 2F,-F, and F,-F, maps, and where a hydrogen-bond could be formed to at least one other polar atom.40 Water molecules were refined as fully occupied oxygen atoms. The progress of refinement is summarized in Figure 3.

Refinement of the structure of the selenomethionyl variant of PBGD was initiated with the final atomic model of the native protein (described herein) but incorporating structural changes indicated by a (Fo,semet-Fo,native) difference Fourier map. l9 Sulphur atoms of methionine side-chains were replaced by selenium atoms. The dipyrromethane cofactor was rebuilt according to an omit map derived from the coefficients (Fo,semet-Fc'), where F,' and phases were calculated without con- tribution from atoms of the cofactor and of the side- chain of Cys242. In addition, owing to the lower res-

olution of the selenomethionyl data set, only those water molecules from the native model having an atomic temperature lower than 50 11' were included (150 of 249). The refinement proceeded through 20 cycles of restrained least-squares (PROLSQ).

During all refinements the five atoms of the pyrrole ring, plus the first extra-planar atom of each side-chain substituent in each porphobilinogen moiety of the dipyrromethane cofactor, were restrained to be in the same plane. However, no restraints have been imposed on the two dihedral angles around the bridging methylene group.

The current atomic coordinates and structure-factor information have been deposited with the Brookhaven Protein Data Bank.41 Most of the fig- ures were generated using the SETOR suite of programs:' kindly provided by Dr. Stephen Evans.

RESULTS Structural Description The atomic model and quality of the coordinate set

The final atomic model of native PBGD includes residues 3-48 and 58-307, the dipyrromethane cofactor, 249 water molecules and one acetate ion (in total, 2,275 protein and 253 solvent atoms). The crystallographic R-factor and correlation coefficient between F, and F, for all 33,889 X-ray data in the resolution range 10.0-1.76 A are 0.188 and 0.959, respectively (0.173 and 0.963 for the subset of 28,873 reflections with F 2 3aF in the range 6.0-1.76 A). Poor agreement between F, and F, was observed for the very low resolution shell (10-5 A), arising presumably from neglecting the bulk solvent and, for the highest resolution shell (1.8-1.76 A), reflecting the large number of weak structure-factor amplitudes. The Luzzati analysis43 estimates 0.20 as the overall r.m.s. error in atomic positions. As detailed in Table 111, overall deviations from ideal stereochemistry in the PBGD model are reasonably small.

In general, the fit of the atomic model to electron- density maps is very good (see Fig. 4). Apart from a strong peak, likely to be due to oxidation of the dipyrromethane cofactor, as discussed below, there are no features with height greater than + 0.5 e/A3 in the final F,-F, difference map. No well-defined density can be observed for residues 1-2 and 308- 313 at the polypeptide chain termini or for residues 49-57 and for the side-chains of several solvent-accessible residues. It is predicted that residues 49-57 form a mobile loop in an exposed position at the front surface of the molecule. The high accessibility and mobility of this segment of polypeptide chain is consistent with the occurrence both of nearby sites of chymotrypsin and trypsin sensitivity, at Phe62 and Lys64, respectively. Two of the most readily py- ridoxylated lysines in the E . coli protein are found in this loop at residues 55 and 59.44

G.V. LOUIE ET AL.

0.50

0.40

0.30

0.20

0.10 0 10 20 30 40 50 60 70 80 90 100 110

Refinement cycle

Fig. 3. Progress in the 14 rounds of refinement of the structural model of wild-type PBGD. Each refinement cycle represents a run of restrained least-squares, except for one run of X-PLOR at the start of round 4. The atomic model was inspected against

The selenomethionyl variant of PBGD1’ has an R-factor of 0.221 for all 19,542 reflections in the resolution range 10-2.0 A. The atomic model for this protein differs very little from that for the native PBGD, the overall r.m.s. positional deviations being 0.13 for main-chain atoms and 0.17 A for all protein atoms except for the dipyrromethane cofactor, as discussed below. Therefore, unless otherwise indicated, the structural descriptions that follow apply to both the native and selenomethionyl variant PBGD, although some of the quantitative details are derived from the higher resolution native structure.

Polypeptide chain conformation Description of topology and secondary structure.

The PBGD molecule has approximate overall dimensions of 57 x 43 x 32 A. The polypeptide chain of 313 amino-acid residues is folded into three alp domains of approximately equal size, as illustrated schematically in Figure 5. Domain 1 (residues 3-99 and 200-217) and domain 2 (residues 105-193) have a similar overall topology, namely, a doubly-

electron density maps after each round. Comments indicate points at which the X-ray reflection data set was changed or at which notable revisions to the atomic model were made. See Experimental Methods for further details.

wound, parallel p-sheet of five strands. In each sheet, there are four parallel and one antiparallel strands (domain 1: - l x , + 2 x , + 2 x , -1; domain 2: -2, -1 x , +2 x , + 2 x ).45 The initial wind contains two ap units, whereas the second wind contains one ap unit into which the antiparallel p-strand is inserted. The a-helical segments pack against each face of the sheet and are oriented essentially parallel to the P-strands. The polypeptide- chain segment connecting strands, p3, and p4,, contain two conserved proline residues and forms no regular structure, although an a-helix might be expected. The C-terminal domain 3 encompassing residues 222-307 is an open-faced, three-stranded antiparallel p-sheet (+ 1, + 1). Covering one face are three a-helices, one of which precedes and two which follow the p-meander.

Domains 1 and 2 can be considered to result from the duplication of a motif comprising a sheet of four parallel (3-strands with intervening a-helices which is followed by an additional separate strand that is also oriented parallel to the preceding four strands.

STRUCTURE OF E. COLI PBGD

TABLE 111. Agreement With Ideal Stereochemistry in the Atomic Models for Wild-Type and Selenomethionyl Variant PBGD

RMS RMS deviation deviation Refinement from ideal from ideal target

Stereochemical class native wild type weighting

1-3 angle distance (A) 0.046 0.039 0.035 1-4 planar distance (A) 0.065 0.054 0.050

Chiral centres (A2) 0.195 0.153 0.150

1-2 bond distance (A) 0.020 0.016 0.020

Planes (A) 0.021 0.013 0.020

55

Non-bonded contacts (A) Single-torsion

Multiple-torsion

Hydrogen bond

0.177 0.170 0.250

0.168 0.160 0.250

0.187 0.184 0.250

(-0.300)*

(-0.100)*

(- 0.200)* Staggered (?60", 180") side-chain torsion angle ("1 14.7 17.5 15.0 Planar (0' or 180") torsion angles (") 3.6 2.2 3.00 Temperature factors (A,) 1-2 bond (main chain) 1.87 1.26 1.80 1-3 bond (main chain) 2.61 2.00 2.00 1-2 bond (side chain) 3.35 2.47 2.00 1-3 bond (side chain) 5.30 3.68 2.50

* For non-bonded contacts, the rms deviations from ideality incorporated the indicated reduction from the sum of the radii of the two atoms involved in contact.

Fig. 4. Typical electron density from a 2F0-F, map for wild-type oxidized PBGD. The region of the molecule shown is the antiparallel p sheet in domain 3. The contour level is 2.3 times the overall r.m.s. level.

The duplicated motifs are then related by an approximate two-fold (163") axis. The isolated strand of each motif interdigitates into the parallel @-sheet of the dyad-related motif to become the single antiparallel strand, thus yielding the mixed @-sheet in each domain. The approximate two-fold axis passes a t -45" to the propeller-twist axis of each of the @-sheets. A point of note relating to protein folding

is that in domain 1 the antiparallel strand (@5,) occurs along the polypeptide chain only after the en- tire domain 2 is completed and may, conceivably, intercalate into an existing four-stranded parallel sheet.

Superp~s i t ion~~ of the two domains, as depicted in Figure 6, yields an r.m.s. positional discrepancy of 1.7 A for 68 pairs of equivalent Ca-carbons. Both the


Fig. 5. Ribbon representation4* of the polypeptide backbone and cofactor dipyrrole of wild-type oxidized PBGD. p-Strands are represented as broad arrows, a-helices as ribbons, and coil regions as thin rope. Side-chains forming direct interactions with the cofactor are drawn with lines of medium thickness.

strands and helices are shorter in domain 2 and there is little sequence similarity, except around the first a-helix in each domain (Serl3 = Ser129, Gln19 = Gln135, and Pro32 = Prol41). Interestingly, a P-bulge in the fourth p-strand of domain 1 occurs also at the corresponding position in domain 2.

Znterdomin connections. The two polypeptide chain connections between domains 1 and 2, which arise from the duplication of a motif having a core four-stranded sheet along with an additional strand inserted into the other domain (see above), are formed by residues 100-104 and 194-199. These “hinge” segments run roughly antiparallel to one another and interact through a number of hydrogen- bonds (102 N-199 0, Gln198 NE2-104 0, Arg 101 NH1-198 0). Each segment directly connects the C-terminal edge strand of the origin domain (P4,) to the antiparallel strand of the opposing domain (PEi2). The series of polypeptide-chain segments P4,, P!j2, . . . , P42, and P5, can be considered to form a pair of adjacent antiparallel P-strands. The overall topology of this unit is consistent with the normally observed right-handed twist along each even though the central portion of each “strand” lacks typical P-sheet-type conformation and hydrogen- bonding. Domains 1 and 3 are joined by a single, short segment of polypeptide chain extending from residues 218-221.

Secondary structural elements. The polypeptide-

chain backbone hydrogen-bonding in PBGD is shown in Figure 7. The average main-chain dihedral angles for each class of regular secondary struc- t ~ r e ~ ~ are typical of those observed in other highly refined protein structure^.^' Overall, approximately 38% of the residues have been assigned a helical conformation, 25% extended, 8% turn, and 29% coil.

A number of minor irregularities in the helical segments and in local capping interactions are observed.48 Within helix a12, two side-chains (Gln135 and Arg139) form hydrogen-bonds to the backbone of the preceding turn of the helix, thus disrupting regular hydrogen-bonding. Helix al, has a marked curvature in a plane roughly parallel to the face of the p-sheet against which the helix is packed. This curvature is likely to result from intra-helical hydrogen-bonding by two threonine side-chains (Thr229 OG1-225 0 and Thr236 OG1-232 0) that, in both cases, lengthen the hydrogen-bond formed by the amide nitrogen of the succeeding residue.

Five of the a-helices are terminated by an ac2 d i s t ~ r t i o n , ~ ~ in which the second residue (usually) following the helix has an aL conformation. For four of the six 3,,-helices, the succeeding residue has backbone angles of very nearly (-73”, 125”), which directs its carbonyl group perpendicular to the helix axis pointing towards the solvent and directs its side-chain towards the interior of the molecule. The edge strand P4, of the domain-1 P-sheet contains a

STRUCTURE OF E. COLI PBGD 57

93

Fig. 6. Superp~sition~~ of the a-carbon backbones of domains 1 (thick lines) and 2 (thin lines) of oxidized PBGD. The superposition is effected by a 162" rotation about the indicated axis (dashed line). For reference, the dipyrromethane cofactor, correctly positioned with respect to domain 1 in this figure, is shown, and every tenth a-carbon is labeled.

classic P - b ~ l g e ~ ~ at residues 96 and 97 (96,97;203). In addition, a similar distortion 184,185;108 occurs a t the corresponding site in strand P42 of domain 2.

Reverse turns and hairpins. The polypeptide chain of PBGD forms 12 reverse turns. Most occur in the loops connecting secondary structural elements. However, two overlapping type-I1 turns (residues 195-198 and 197-200) constitute much of the second hinge-segment, with the amide nitrogen of the central peptide-bond of the 197-200 turn forming a hydrogen-bond to the propionate side-group of the dipyrromethane cofactor (C1 ring). The turn at residues 31-34 may be disordered between type-I and type-11, since electron density maps indicate that the peptide-bond unit between the second and third residues (Pro32 and Gly33) can adopt either of the two opposite orientations. There are two PIP-hairpins within the p-meander in domain 3: residues Asp254- Gly255 (class with a type-I' reverse turn) and residues Ala265-Pro266-Asp267-Gly268Ser269- Gln270 (class 4:6, within which is a type-I reverse turn). The second contains the invariant Gly268 in an a,-conformation and constitutes the major point of contact between domains 3 and 2.

Interdomain interactions. Apart from the polypeptide-chain connections, there are only a few direct interactions between domains 1 and 2: the hydrogen- bonds Argll NH1-Asnl51 OD1 and Arg149 NH1- Gly45 0, and van der Waals packing of the indole ring of Trpl8 against the aliphatic portion of the Arg176 side-chain. The packing of domain 3 along- side the rest of the molecule is mediated primarily through polar interactions: with domain 1, Arg232 NH1-85 0 and Arg232 NH2-86 0; with domain 2,

salt-bridges Arg260-Glu190 and Arg273-Glu138 and with the interdomain hinge region, 248 N-194 0, 101 N-Thr224 OG1, Glu197 N-Glu231 OE2 and the salt-bridge Arg101-Glu231. Notably, site-directed substitutions at either of the invariant ArglOl or Arg232 reduce enzyme This interdomain region also contains several trapped water molecules (Wat407, 417, 427, 429, 459, and 529), but there are small, hydrophobic interfaces with both domain 1 (Met82, Ile98, Gly197, Ala225, Va1228) and domain 2 (Leu 130, Cys 134, Leu 193 Pro 245, Leu 262, Pro 266, Gly 268).

Conformation of side-chains. In general, for the majority of residues in PBGD, side-chain conformations are consistent with documented prefer- e n c e ~ . ~ ' , ~ ~ The overall r.m.s. deviation of side-chain dihedral angles from optimal values is 14.7". Several side-chain groups, as judged by their appearance in electron density maps, occupy at least two discrete positions: Lys26(NZ), Arg74(NE, CZ, NH1, NH2), and Ser289(OG). Leu136 (CG, CD1, CD2) and Ile257 (CD1) have alternative packing arrangements in the core of the protein. The highly conserved Va1196, that occurs within the second-hinge strand at the rear of the active-site cleft of the enzyme, appears to have two equally populated conformers.

Dipymmethane cofactor Active-site cleft. The active-site cleft containing

the dipyrromethane cofactor4 occurs at the interface between domains 1 and 2 (see Fig. 5). The floor and ceiling of the cleft are formed mainly from the C-termini of the p-strands, the N-termini of a-helices, and the connecting loops. One face of the a3, helix


Fig. 7. Schematic of main-chain hydrogen-bonding in the oxidized form of PBGD. Residues forming hydrogen-bonds within or at the ends of regular secondary-structural elements are outlined by ellipses (for helices) or rectangles (for p-strands). Hydrogen- bonds are drawn as arrows pointing from the residue with the

amide nitrogen to the residue with the partner carbonyl oxygen. The inset at the top right shows a schematic of polypeptide chain topology, and the nomenclature of the secondary structural elements (p-strands are represented as broad arrows, and a-helices as cylinders).


Fig. 8. Hydrogen-bonding interactions formed by the cofactor binding loop (residues 239-245). Main-chain atoms are drawn with thin lines, side-chain atoms with medium-thickness lines, and the cofactor dipyrrole (oxidized form) in thick lines. Water molecules forming bridging interactions between the loop and the body of the molecule are drawn as small crosses. Hydrogen-bonds are drawn as thin dashed lines.

and the hinge segments linking the two domains constitute the rear wall of the cleft. The cleft, with dimensions of - 15 x 13 x 12 A, spans approximately half of both the width and depth of the molecule. The positioning of the cofactor near the entrance to the cleft leaves a considerable volume of internal space vacant, in which many well-ordered water molecules are bound.

Cysteine 242. Cys242, which covalently attaches the dipyrromethane cofactor to the protein, is the second residue of a type-I p-turn situated at the end of the loop joining al, and p1, in domain 3. This loop projects into the cleft between domains 1 or 2. How- ever, there are no extensive direct interactions with either domain; the only interactions are made through bridging water molecules (Fig. 8). The thioether linkage to the cofactor adopts a reasonably favorable "left-handed spiral"45 conformation, very similar to that of the thioether between the haem group and Cysl4 in cytochrome c.52 In the oxidized protein, the two dihedral angles about the Sy atom have values of -80" and -62", and X1 of the Cys242 side-chain is -60". These values in the reduced selenomethionyl variant differ slightly (- 78", -75", and -5T, respectively).

Cofactor oxidation state and conformation. There is well-defined electron density for the cofactor dipyrrole system in both the oxidized and reduced wild-type PBGD and in the reduced selenomethionyl variant (Fig. 9). Two distinct conformations of the dipyrrole are observed. Both conformers occur in the

wild-type protein, where the predominant conformer (80% occupancy) appears to represent the oxidized form of the dipyrrole (see discussion below) with the minor conformer (20% occupancy) representing the native (reduced) dipyrromethane. This latter conformer occurs a t essentially 100% occupancy in the selenomethionyl variant that was crystallized under reducing conditions.

The reduced dipyrromethane and the oxidized dipyrrole differ in the conformation about the bridging meso-carbon. However, in both cases the conformation adopted, with the CHB-ClB bond grossly trans to the C3A-C4A bond, the interaction between the ring C2 pyrrole and the propionate group of ring C1 is minimized and the two pyrrole nitrogens are adjacent at one edge, whereas all four acidic side- groups radiate from the other edge (see Fig. 10b for atom nomenclature). In the oxidized dipyrrole, the two dihedral angles (as defined by the atoms C3A- C4A-CHEClB-C2B) about the bridging methylene group are 171" and -173"; thus, the pyrrole rings have an interplanar angle of 11" (considering each pyrrole-ring plane to be defined by the five atoms of the pyrrole together with the first atom of each of the side-groups). In the reduced dipyrromethane system, both dihedral angles (120" and -114") are -60" less obtuse and thus the two pyrrole rings have an interplanar angle of 59" and form a distinct elbow at the methylene group.

With both conformations of the dipyrrole system, the four pyrrole P-side-chain substituents have the


9

Fig. 9. Electron density for the cofactor dipyrrole in its (a) oxidized and (b) reduced selenomethionyl variant forms. Electron density is from an Fo-F,’ map where values of F,’ were calculated with all atoms of the dipyrrole and the Cys242 side-chain omitted. The contouring levels shown are 3.4 and 10.1 times the rms. level (u) in (a), and 3.1 and 12.3 u in (b).

bond between the first and second atoms lying nearly perpendicular to the plane of the pyrrole ring. However, for the propionate group of ring C1 and the acetate of ring C2, the dihedral angle about this bond differs by -180“ between the reduced and oxidized conformers (see Fig. 10). In addition, the two propionate side-groups in the oxidized conformer have essentially the most sterically favored, fully extended conformations, as does the ring-C1 propionate in the reduced conformer.

The positioning of the dipyrrole in its reduced (native) and oxidized conformers is compared in Figure 10. The rings C1 and C2 are both positioned more deeply within the active-site cleft in the reduced (native) form. The small rearward shift of ring C1 (by -0.7 A) arises from a small upward shift of Cys242 (by -0.3 b) and the slightly altered conformation about the thioether linkage, as described above. The more substantial rearward shift of ring C2 (by -3.4

A) is largely due to the altered conformation of the bridging methylene group of the reduced dipyrromethane form. These structural differences are entirely consistent with the observed difference-density peaks in the (Fo,Semet-Fo,native) Fourier map.19

It should also be noted that in the native reduced protein, the strongest electron density for the internal positioning of ring C2 occurs at sites corresponding to the two carboxylate groups. With the C2 ring in its more external, oxidized position these sites can instead be occupied by solvent molecules. In the current atomic model, a free acetate ion and a water molecule (Wat401) have been included. Similarly the site occupied by the propionate carboxylate in the oxidized native protein is found to bind a free acetate ion in the reduced selenomethionyl variant.

Cofactor interactions. The acetate and propionate side-groups of the dipyrromethane form the majority of the cofactor-protein interactions, consistent with

STRUCTURE OF E . COLI PBGD 61

0 1 B

A P

A

dipyrromethene dipyrromethane

Fig. 10. a: Comparison of the conformations of the cofactor dipyrrole in its oxidized [-thick line] and reduced selenomethionyl variant [-thin line] forms; b: nomenclature for atoms of the dipyrromethane cofactor, and chemical structures of the possible oxidized forms of the dipyrromethane.

the inability of the apoenzyme to synthesize the cofactor from the porphobilinogen analog 2-amino- methylpyrrole5 (in which the side-substituents are replaced by hydrogen atoms). These acidic pyrrole side-chains are involved in extensive salt-bridging and hydrogen-bonding with the surrounding protein matrix and with bound water molecules (Fig. 11). Each of the carboxylate-oxygen atoms (except 01B in the oxidised native protein) is involved in two or more interactions. The participating polypeptide- chain functionalities are: arginyl guanidiniums (Argll, Argl31, Arg132, Arg149, and Arg155),

seryl and threonyl hydroxyls (Serl3, Ser81, Thr127, and Ser129), backbone amide nitrogens (128 N, 150 N, 152 N, 170 N, and 199 N) and additionally the amino function of Lys83. The side-chain groups, which are largely invariant, take part in further hydrogen-bonding with adjacent protein and solvent atoms. Notably, three of the four carboxylate groups of the cofactor are positioned very near the N-termini of a-helices: both the acetate and propionate groups of ring C1 are slightly displaced from the axis of al, and the propionate group of ring C2 in the native, oxidized protein occurs at the juxtaposi-


tion of the ends of all and a2, and in the reduced selenomethionyl variant near the end of a3,. The interactions formed by the pyrrole rings themselves include hydrogen-bonds from both pyrrole nitrogens to the side-chain carboxylic-acid group of Asp84 and also the stacking of pyrrole ring C2 against the phe- nyl ring of Phe62 in the native oxidized protein. The involvement of the cofactor, both in forming an extensive network of interactions which cross-link the three domains and in partially neutralizing the considerable electropositivity within the active-site cleft, satisfactorily explains the more stable and compact tertiary structure of the holoenzyme as compared to the ap~enzyme.~

Of the two porphobilinogen units of the cofactor, the first (the C1 ring that is bonded to Cys242) forms a greater number of specific interactions. The tighter binding of the ring C1 is consistent with the considerably lower atomic temperature factors of this group (17.9 A" as compared to 22.7 A" for ring C2 in the oxidized conformer and 17.7 A" as compared to 22.4 A" for ring C2 in the reduced conformer) and also with the less disparate positioning of the ring C1 unit (and particularly the side-substituent carboxylate groups of this ring) between the reduced and oxidized conformers (see Fig. 10).

The cofactor-protein interactions observed in PBGD bear some resemblance to those occurring in the C-phyc~cyanins.~~ These proteins also carry an open-chain, polypyrrolic prosthetic group (a tetrapyrrolic bilin) also covalently bound through a thioether bond to a cysteine side-chain. In particular, the central two pyrrole units of the bilin each bear one propionate side-substituent and together have a conformation very similar to that of the dipyrrole in the native oxidized form of PBGD. The nitrogens of these two pyrrole rings both hydrogen- bond to an aspartate side-chain from the protein and the propionate groups form salt-bridges with arginine and lysine side-chains. In contrast, in the bilin binding the two propionate groups of the noncovalently-bound bilin are directed into the solvent. Thus the polypeptide-chain interacts with the polypyrrole mainly through van der Waals contacts between atoms of non-polar protein side-chains and atoms of the pyrrole rings.

Evidence for the nature of the oxidation of the dipyrromethane in PBGD. There are several indica- tions that, as discussed above, the predominant conformer (80%) of the dipyrrole in the wild-type PBGD represents an oxidized form. First, although the native reduced dipyrromethane is not a chromophore, the crystals typically have a deep-yellow color. The coloration of the crystals intensifies with time, but its development can be largely inhibited by the presence of a reducing agent. Thus the crystals of the selenomethionyl variant, that contain exclusively the native dipyrromethane, were essentially color- less. Second, an excess lobe of electron density con-

nected to the free a-position of ring C2 (see Fig. ga) is consistent with the presence of a carbonyl-oxygen atom. Third, the observed near-coplanarity of the two rings of the cofactor in the oxidized conformer may signify the presence of a double bond within the bridging methylene group. These considerations point to an oxidized form of the dipyrromethane such as a dipyrromethenone. Notably, dipyrrome- thenones are strongly chromophoric (wavelength,, = 390nm) and undergo a light-induced, internal cis- trans isomerization, properties consistent with the color and light-sensitivity of the ~rysta1s.l~ The carbonyl oxygen of a dipyrromethenone would be appropriately positioned to accept a hydrogen-bond from the carboxylic-acid function of the Asp84 side- chain. However, the electron-density maps cannot rule out the presence of other forms of the dipyrrole. These include a dipyrromethene, that lacks the carbonyl oxygen and a dipyrromethanone, in which the C1B atom is sp3-hybridized and as a consequence the C1B-CHB bond is out of the plane of the pyrrole ring C2. A mixture of some, or all, of these oxidized forms is the most probable situation. Although the dipyrrole may exist in an oxidized form, it must be emphasized that both porphobilinogen units adopt apparently favorable conformations and form numerous stabilizing interactions with the protein molecule. Therefore, it is quite likely that the more external site occupied by the C2 ring in the oxidized form in fact corresponds approximately to the binding site of the substrate porphobilinogen (see discussion below).

Hydrogen- bonding Both the average geometric characteristics ob-

served for the three classes of hydrogen-bonds (main-chain + main-chain, main-chain + side- chain, and side-chain + side-chain) and the high degree of saturation of hydrogen-bonding potential for PBGD agree well with observations from other protein structure^.^' Intramolecular hydrogen- bonds are formed by 66.8% of polar main-chain atoms and 48.8% of polar side-chain atoms.

As discussed above, within all secondary structural elements, main-chain hydrogen-bonding is quite extensive (see Fig. 7) and largely regular, with hydrogen-bond energies clustering fairly tightly (standard deviation 0.6 kcal/mol) about an overall mean of 2.1 kcal/mol for 176 hydrogen-bonds. Rela- tively little main-chain hydrogen-bonding occurs outside of these elements and the reverse turns.

Considering both intra- and intermolecular hydrogen-bonding, 88.2% of all protein (main-chain and side-chain) polar atoms participate in hydrogen- bonds. The high degree of hydrogen-bonding (overall saturation 75.3%) probably contributes significantly to the considerable thermal stability of the folded state of the protein molecule.


ai

Fig. 11. Schematic representation and stereo view of the hydrogen-bonding network around the cofactor dipyrrole in its (a, i, ii) oxidized and (b, i, ii) reduced selenomethionyl variant forms. In the stereo views, main-chain atoms are drawn with thin lines,

side-chain atoms with medium lines, and the cofactor with thick lines. Hydrogen-bonds are shown as dashed lines. Water molecules within the active site cleft are drawn as small crosses.

Hydrophobic cores The PBGD molecule contains three distinct hydro-

phobic cores, one in each of the three domains. These cores are formed almost exclusively from the inter- digitation of side-chains projecting from the faces of the P-sheets and from the flanking a-helices. The majority of the residues involved are aliphatic and highly conserved. The most commonly observed substitutions are by residues with side-chain hydroxyl groups. Somewhat unusually, PBGD contains very few aromatic residues, and thus these contribute little to forming the hydrophobic cores. In domain 3, the hydrophobic core includes the interface between the two longer helices, which are positioned side-by- side (with an interaxial angle of -30") against one

face of the core P-sheet. The interhelical interface is formed mainly by a number of residues with small aliphatic side-chains (Ala230, Ala233, Ala283, Gly287, and Ala291).

Typical of most globular proteins, the interior of the protein molecule is tightly packed. Analysis by the method of C ~ n n o l l y ~ ~ reveals only two small, internal cavities large enough to accommodate a probe sphere 1.4 A in radius. One, located in domain 1, is lined by the side-chains of Va163, Met82, Va185, Pro86, Phe89, Leu95, and Glu204, as well as the face of the His80 imidazole ring, and reflecting its predominantly hydrophobic nature, is without a bound water molecule. The other cavity, in domain 2, is delimited by Leull6, Leu136, Ile168, Leu187, and


bii

bi

/ /

Fig

Ile191, as well as the side-chains of Arg139 and Ser192 and the carbonyl group of Arg132. This cavity c0ntain.s two water molecules (442 and 535), hydrogen-bonded to these polar groups.

Ion-pairs PBGD forms an unusually large number of in-

tramolecuiar-ion-pairs (261, (see Table IV), more than twice that expected for a protein of 313 resi- d u e ~ . ~ ~ Arginine plays a major role in ion-pair formation, as 17 out of a total of 27 arginine residues in E . coli PBGD participate and all but four ion-pairs involve an arginyl guanidinium group. Examples of all types of twin N-twin 0, twin N-single 0, and single N-single 057 guanidinium-carboxylate interactions are observed. Notably, in all of the twin

Gly240

His80

. l l b .

N-twin 0 interactions (including four instances of the rarer type 2), the guanidinium and carboxylate groups are nearly coplanar. As described above, interactions between the protein and the acidic side- groups of the cofactor account for seven of the sev- enteen ion-pairs. In addition, a number of the ion- pairs occur buried within the protein interior with Arg101-Glu231, Arg260-Glu190, and Arg273- Glu138 interactions occurring between domains. These interactions may be of particular importance in stabilizing the protein tertiary structure.

Temperature factors The spatial distribution of main-chain tempera-

ture factors is shown in Figure 12. Clearly the most rigid regions of polypeptide chain correspond to the

STRUCTURE OF E. COLI PBGD

TABLE IV. Intramolecular Ion-Pairing in PBGD*

65

Interatomic No. Interacting atoms distance (A) Notes 1 Arg7:NE Asp76:OD2

Arg7:NHl Asp76:ODl 2 Argl1:NE Asp46:OD2

Argl1:NHl Asp46:ODl 3 Lys64:NZ Glu67:OEl 4 Arg74:NH2 Glu72:OEl 5 His80:NE2 Glu204: OE 1 6 ArglO 1 :NH 1 Glu23 1:OE2

ArglOl:NH2 Glu23 1 : OE 1 7 ArglO5:NE Aspl03:ODl

ArglO5:NHl Asp103:OD2 8 Argl31:NHl Asp 106: OD 1

Argl3 1:NH2 Asp106:OD2 9 Arg132:NE AsplO6:ODl

Arg132:NHl AsplO6:ODl 10 Argl40:NE Asp142:ODl

Argl40:NHl Asp142:OD2 11 Lysl75:NZ Glu102:OE2 12 Arg182:NHl Asp160:OD2

Arg182:NH2 Aspl6O:ODl 13 Arg184:NH2 Asp1 14:OD2 14 Arg206:NHl Asp76:ODl 15 Arg206:NHl Asp209:OD2 16 Arg237:NE Glu292:OEl

Arg237:NHl Glu292:OEl 17 Arg260:NH2 Glu190:OE2 18 Arg273:NHl Glu138:OEl

Arg273:NH2 Glu138:OE2 19 Arg276:NHl Glu293:OEl

Arg276:NH2 Glu293:OEl Involving DPM cofactor (native protein): 20 Argll :NH1 Dpm:O3B

Argll :NH2 Dpm:03B 21 Lys83 :NZ Dpm:OlA 22 Argl31:NE Dpm:02A

Argl31:NHl Dpm: 0 1A 23 Arg132:NH2 Dpm 03A 24 Argl49:NE Dpm: 0 1B

Argl49:NHl Dpm:02B 25 Arg155:NHl Dpm:OlB 26 Arg155:NHl Dpm:04A

Arg155:NH2 Dpm:04A Involving DPM cofactor (selenomethionyl variant): 20 Lys83 :NZ Dpm: 0 1A 21 Lys83 :NZ Dpm:O2B 22 Argl31:NE Dpm:O2A

Argl31:NHl Dpm:OlA 23 Arg132:NH2 Dpm:O3A 24 Arg132:NHl Dpm:O2B

Arg132:NH2 Dpm:02B 25 Arg155:NHl Dpm:04A

Arg155:NH2 Dpm:04A *Ion-Pair types for arginyl guanidinium-carboxylate interactions are as defined by Singh et aL5'

a-helices and @-strands and also the hinge segments between domains 1 and 2. The loops connecting the regular secondary structural elements are more flexible, particularly those at the periphery of the

molecule. In addition, both the N- and C-termini of the polypeptide chain and the segment containing residues 47 through 60 are extremely flexible, reflecting their highly exposed and relatively uncon-

3.11 2.89 3.07 2.63 3.14 2.77 2.82 3.40 2.67 2.90 2.79 3.39 2.84 3.17 2.75 2.93 3.06 2.89 2.99 2.84 3.05 3.10 3.20 2.85 3.42 2.85 2.82 2.96 2.99 2.79

3.32 3.03 2.90 2.87 3.14 2.84 2.77 3.07 2.95 2.90 3.00

2.90 2.90 2.87 3.14 2.84 2.77 3.07 2.90 3.00

Type 1

Type 1

Type 5

Type 2, buried

Type 1

Type 2, buried

Type 9, buried

Type 1

Type 2

Type 5 Type 4 Type 4 Type 9

Type 5 Type 2

Type 8

Type 8

Type 1

Type 5 Type 1

Type 4 Type 8

Type 1

Type 5 Type 8

Type 8


PBCD temperature factors PBCD temperature factors

Fig. 12. Main-chain temperature factors mapped on the tertiary structure! of native PBGD. For each residue in the polypeptide chain, the a-carbon is drawn with a radius representative of the average main-chain temperature factor. Radii in the range 0.1-1 .O A correspond linearly with the temperature-factor range 11.5-

40.0 A*. The twelve residues with an average main-chain temperature factor greater than 40 A' are drawn with doubled spheres, for which the outer radii range from I .O-2.0 A corresponding to the temperature factor range 40.0-71.8 A'.

strained positioning on the surface of the molecule. For a small number of residues where the side- chains possess lower temperature factors than the backbone, the side chains are usually packed within the hydrophobic core of the molecule.

Solvent structure The majority of the water molecules associated

with a single molecule of PBGD populate the first solvation layer of the protein molecule; 92% of the 249 water molecules are positioned within 3.6 A of a protein atom and the overall average distance to the nearest protein atom is 3.0 A. The temperature factors of the water molecules range from 14.9 to 80.8 A", with a mean of 45.2 A".

The 249 water molecules form a total of 560 hydrogen-bonds, or 2.2 each on average. The characteristics of these hydrogen-bonds are, in general, typical of those found in other high-resolution protein struct~res.~' The majority (89.2%) of the water molecules interact directly with the protein molecule, through a total of 430 hydrogen-bonds (1.94 per water molecule on average). Of the protein-water hydrogen-bonds, 51% involve main-chain atoms and 49% side-chain atoms. As noted previ~usly,~' the

hydrogen-bond distances from water oxygens to oxygen atoms of the protein are shorter on average than those to nitrogen atoms, however, the prepon- derance in number of the former class over the latter (1.44:l overall) is less marked than observed in other proteins.

Several water molecules are located within the interior of the PBGD molecule, including the interface between domain 3 and the other two domains. Most serve to satisfy the hydrogen-bonding require- ments of buried main-chain amide and carbonyl groups and have much lower than average temperature factors. These water molecules probably have an important structural role. In addition, a large number of water molecules are sequestered within the active-site cleft of the enzyme, behind the dipyrromethane cofactor.

There is a clear trend for water molecules forming the greatest number of hydrogen-bonds to have the lowest atomic temperature factors. The most tightly bound water molecules are those that interact directly with the protein molecule and, in particular, with the polypeptide-chain backbone. This good agreement with expected trends substantiates the meaningfulness of the refined water structure.

STRUCTURE OF E . COLZ PBGD 67

Crystal packing The packing of molecules of PBGD within the

P2,2,2 crystal lattice is shown in Figure 13a,b. The centering of PBGD molecules very near the 2,-screw axes running parallel to b at z = 1/2 gives rise to columns of molecules arranged end-to-end (involving seven hydrogen-bonds, one salt-bridge, and 10 van der Waals contacts at the interface; (1/2-x, 1/2 + y, 1 -z)) along these 2,-axes. Adjacent columns of molecules come in contact (via six hydrogen- bonds, one salt-bridge, and three van der Waals contacts; (-x, -y, z)) at the two-fold rotation axes at (z = 0, y = 0) and (z = 1/2, y = 1/21, thus generating a layer of thickness c (50.5 A) oriented parallel to the ab plane, within which the most important intermolecular interactions are formed. consistent with the slowness of crystal growth in the c direction (i.e., normal to the large face seen in the external morphology), interactions between adjacent layers are relatively few and are generated by the 2,-screw axes parallel to b a t z = 0 (two van der Waals contacts; (1/2-x, 1/2 +y, -z)). The crystal packing gives rise to two sets of sizeable solvent channels, one running parallel to the columns of molecules described above, along (x = 0, z = 1/8) and (x = 1/2, z = 7/8) and the other running perpendicular, along the two- fold rotation axes at (x = 112, y = 0) and ( x = 0, y = 1/2). This is of importance to experiments involving the soaking of substrates or inhibitors into crystals since the active-site cleft of a PBGD molecule in the crystal lattice is accessible from the first set of solvent channels.

There are two intermolecular hydrogen-bonding interactions involving a pair of acidic side-chains (Glu37 and Asp142, and Glu72 and Glu292). In each case, at least one of the interacting carboxylic-acid groups must be protonated, which may explain the requirement of a pH below -5.5 for crystal formation. Removal of these acid-acid side-chain interactions through site-directed mutagenesis may facili- tate crystal growth of PBGD at a pH closer to 8.5, the pH optimum of the enzyme.

Amino Acid Conservation in the PBGDs Sequence alignments

An alignment of the known amino-acid sequences of PBGD, from E. ~ o l i , ~ ~ man,59 rat,60 mouse,61 Ba- cillus subtilis,62 Euglena g r a ~ i l i s , ~ ~ yeast,64 Arabi- do psi^,^^ Pseudomonas aeruginosa,66 pea,67 Clostridium josui,68 Chlorobium ~ibrioforme,~' and Mycobacterium leprae7' is shown in Table V. Se- quence identities between individual pairs of proteins range from 32% to 96% and between E. coli PBGD and each of the other proteins from 39% to 66%.

The majority of the insertions and deletions in the sequences of the other species of PBGD relative to the E. coli protein are short, and with one possible

exception, all occur at surface loops. The two higher plant sequences are shortened at the C-terminus, after the end of well-ordered a-helical structure (a3,) in the E . coli protein. The long 29 residue in- sertion in the animal sequences probably occurs in the loop following strand p-33 in domain 3, and is thus positioned quite distant from the active-site cleft of the molecule. In the P. aeruginosa protein, a glycine may be inserted between residues 26 and 27, thus causing a bulge within helix all (Fig. 7 inset).

Invariant residues The majority of the invariant residues (Table VI)

are clustered immediately around the active-site cleft (Fig. 16). These are involved in the catalysis of the bond-making reactions, in forming direct interactions (particularly salt-bridges and hydrogen- bonds) with the dipyrromethane cofactor (and also with the porphobilinogen substrate) and possibly in accommodating the growing polypyrrole product. Other invariant residues from the surrounding protein matrix participate in forming a further extensive network of hydrogen-bonding that may be of importance in stabilizing the conformations of the groups interacting directly with the cofactor (see Fig. 8). The high degree of invariance of residues in the vicinity of the active-site cleft indicates that the mechanism of the E. coli enzyme probably holds also for the PBGDs from all other species.

One unexpected amino-acid substitution is glycine in the P. aeruginosa protein for (the otherwise invariant) Argl31. Argl31 forms an important salt- bridge to the acetate group of cofactor ring C1. His- tidine16 or leucine15 replacement, through site-directed mutagenesis in the E. coli protein, completely prevents assembly of the cofactor and glutamine replacement in humans, and gives rise to an unstable protein and the acute intermittent porphyria disease state.3 In the P. aeruginosa protein, however, a functionally equivalent arginine may be provided by a compensating arginine replacement of the nearby Ser129.

More remote from the active-site cleft, many of the invariant residues appear to have roles in stabilizing the protein fold. In particular, some promote turn conformations, contribute to the hydrophobic core of the molecule, form capping interactions at the ends of helices or form structurally important hydrogen-bonds or ion-pairs. Additionally, sites of restricted steric space are typically occupied by glycine or alanine residues and, at several positions in the polypeptide chain, a conformation inaccessible to residues bearing a side-chain is adopted by an invariant glycine.

Other surprising mutations occur in recently reported sequences, including G268S,6' D106R R101V,69 V263C: A200I: L116Q.70 These are listed as otherwise invariant locations in Table VI and re- arrangements of local structure are required to ac-


Fig. 13. Molecular packing in crystals of PBGD. a: View parallel to the crystallographic c-axis. Shown is a portion of two adjacent columns of molecules arranged along the 2, screw axes that run parallel to the b-axis and lie in the z = 4 plane. Within the portion of the a6 layer shown, the molecule MI (drawn in bold lines) makes contacts with the two molecules M2 and M3 related by the 2, axis and with the molecule M4 related by the 2-fold

commodate them. The substitution D106R is of particular note in view of the role of D106 in stabilizing the R131 and R132 side chains and their interaction with the cofactor.

CONCLUSIONS Implications for the Mechanism of Action of PBGD

A thorough understanding of the mechanism of PBGD will be derived only with further detailed structure-function analyses. Structural information on a non-covalent enzyme-substrate or enzyme-in- hibitor complexes and on the covalent intermediates, ES, ES,, ES,, and ES,, will be particularly important in revealing the precise binding sites of

rotation axis at z = iy = a. b: View parallel to the crystallographic a-axis. The two columns of molecules shown are from adjacent ab layers. The molecule MI (drawn in bold) in one layer makes relatively few interactions with molecules M7 and M9 in the other layer. Molecules M7 and M9 are related to MI by the 2, screw axis running parallel to the b-axis in the z = 0 (or z = 1) plane.

the porphobilinogen substrate and the growing polypyrrole chain. However, the existing structural details of the holoenzyme currently allow useful inferences on the mode of action of PBGD to be drawn. These inferences form the basis for the mechanistic models described in this section that are helpful for the design of further experiments, particularly those involving site-directed mutagenesis.

Porphobilirwgen-biding sites within the active-site cleft

The observed dipyrrole-protein interactions in the reduced and oxidized wild-type and reduced selenomethionyl forms of PBGD define at least three binding sites for porphobilinogen moieties within


TABLE V. Alignment of Known Amino Acid Sequences of PBGD With JOY" E. coli Structural Environment Description on Uppermost Line of Alignment (Maligns1)*

51 101 151 201 251 301 351 401 a n v L ~ I A T ~ ~ s p l A l u ~ A h y V k d k L m a s h p g L v v e l v p

E . coli - - - _ - - - - _ - M L ~ ~ ~ ~ L R I A T R Q ~ P L A L F Q A G Y V K D K - L ~ A F G P G L - - - - V ~ E L V P Human - - _ _ - - - - _ - - - M R V I R V G T R K S Q L A R I Q T D S V V A T - L K A S Y P G L - - - - Q F E I I A Rat - - - - - - - - _ - - - ~ R V I R V G T R K S Q L A R I Q T D T V V A M - L K T L Y P G I - - - - Q F E I I A Mouse - - - - - - - - - - - - M R V I R V G T R K S Q L A R I Q T E T V V A M - L K A L Y P G I - - - - Q F E I I A Pea S L A V E Q Q T Q Q N K T A L I R I G T R G S P L A L A Q A H E T R D K - L M A S H T E L A E E G A I Q I V I Arabidopis C V A V E Q K T - - - R T A I I R I G T R G S P L A L A Q A Y E T R E K - L K K K H P E L V E D G A I H I E I Euglena - - - S T T G S N I G A G K T V R V A T R K S P L A M U Q A E F I Q S E - L E R L U P G I - - - - T V E L Q P Bacillus - - - _ - - - - _ - - M M R T I K V G S R R S K L A M T Q T K H V I Q K - L K E I N P S F - - - - A F E I K E Yeast - - - _ - - - - _ - M G P E T L H I G G R K S K L R V I Q S N H V L K L - I E E K Y P D Y - - - - D C K V F T Pseudomom - - _ _ - - - - - - M S S R E I R I A T R Q S R L A L U Q A E Y V N S T G L E Q A H P G L - - - - T V T L L P C. jmui - - - - - - - - M V F D M K K I R I G S R D S K L A I I Q S E L I M S A - I R K Y D P D I - - - - E L E L I T

- - - _ - - _ - _ - - - _ _ - - - - _ _ - ~ ~ - - - - - - - - - ~ - - - - - _ - - - ~ - - - - ~ M N I S L K L C. vrbrioform M. k p m - _ _ _ - - _ _ _ _ _ - _ - M I E I G T R G S L L A T T Q A A L V R D A L I A N G H P A - - - - - - - E L V I

551 501 551 601 b5l 701 751. q, 651 901 ~ v i r ~ i v i r * * * * r * * * q k g l f V h e L ~ v A L l i i i t - - - - A D I A V H ~ ~ k ~ V p v e f p q

E . wli Human Rat MOW Pea Arabidopis Euglena Bacillus Yeast Peeudomonas C. jmui C vrbrioform M. lepme

E . COIL Human Rat MOW Pea Arabidopis Euglena Bacillus Yeast Pseudomonas c. JosUi

C. urbrioform M. l e p m

E . colt Human Rat MOW Pea Arabidopsia Euglena Bacillus Yeast Pseudomonas

C. uibrioforrn M. lepme

c. JmYZ

E . mli Human Rat Mouse Pea Arabidopsis Euglena Bacillus Yeast Pseudomonas

C. uibmform M. lepme

c. JOSUi

M ~ ~ R G D V I L D T P L A K V G G K G L F V K E L E V A L L E N R - - - - A D I A V H S M K D V P V E F P Q M S T T G D K I L D T A L S K I G E K S L F T K E L E H A L E K N E - - - - V D L V Y H S L K D L P T V L P P I S T T G D K I L D T A L S K I G E K S L F T K E L E N A L E K N E - - - - V D L V V H S L K D V P T I L P P M S T T G D K I V D T A L S K I G E K S L F T K E L E N A L E K N E - - - - V D L V V H S L K D V P T I L P P I K T T G D K I L S Q P L A D I G G K G L F T K E I D E A L I N G D - - - - I D I A V H S M K D V P T Y L P E I K T T G D K I L S Q P L A D I G G K G L F T K E I D E A L I N G H - - - - I D I A V H S M K D V P T Y L P E M S T R G D K I L D S P L R K V G G K G L F V K E L E T A L L E N R - - - - S D I A V H S T K D V P M E L P E I V T K G D R I V D V T L S K V G G K G L F V K E I E Q A L L N E E - - - - I D M A V H S M K D N P A V L P E L Q T L G D Q I Q F K P L Y S F G G K A L U T K E L E D H L Y H D D P S K K L D L I V H S L K D N P T L L P E M T S R G D K L L D A P L R K I G G K G L F V K E L E T A L L E G A - - - - A D I A V H S M K D V P M D F P E M K T T G D K I L D K T L D K I E G K G L F V ~ E L D N A L Y N N E - - - - V D I T V H S Y K D N P L E E N P ~ K ~ T G D ~ L L D S P L S K I G D ~ G L F ~ K D I E K H L L A G E - - - - I D L ~ ~ H ~ L K ~ ~ P ~ V R E K V N T A G D Q S - S A S I D S L G - V G ~ F ~ ~ A L R A ~ I E E G C - - - - V ~ A A V H S Y K D L P ~ A ~ D P

951 100( 1051 1101 5151 1201 1251 1301 1351 1401 g ~ q l v T i C e H i i p t ~ n ~ v s ~ n y a s ~ ~ a ~ p a q s i v ~ ~ ~ ~ ~ ~ ; ~ ~ ~ ~ a i ~ ~ p ~ ~ G L G L V T I C E R E D P R D R F V S N N Y - - - D S L D R L P A G S I V G ~ S S L R R ~ C ~ L A E R R P D L G F T I G A I C K R E N P H D A V V F H P K F V G K T L E T L P E K S V V G ~ S S L R R A A Q L Q R K F P H L G F T I G A I C K R E N P C D A V V F E G K F I G K T L E T L P E K S A V G T S S L R R V A Q L Q R K F P H L G F T I G A I C K R Q N P C D R V V F H P K F I G K T L E T L P E K S A V G ~ S S L R R V A Q L Q R K F P N L E T I L P C N L P R E D V R D A F I S L S A - - - A S L A D L P A G S V I G T A S L R R K S Q I L H R Y P S L K T I L P C N L P R E D V R D A F I C L T A - - - A T L A E L P A G S V V G ~ A S L R R K S Q I L H K Y P A L G L V L G V I C K R H D P C D A I V F P K G S N L K S L E D L P H G A R V G T S S L R R Q C Q L L L K R P D L G L V I G C I P E R E D P R D A L I S K N R - - - V K L S E M K K G A V I G T S S L R R S A Q L L I E R P D L

G L G L Y T I C E R E D P R D A F V S N T Y - - - A S L E Q L P A G S V V G T S R L G R Q A Q L L A R R P D L E L P V V A L S K R E D P R D A F I L - - - - - - - P Q N G E N G G E P I G S S S L R R Q L Q L K E L F P G C A U L S P R S P S V K T P R R H H F - - - - Q V R Q G V D G P S A E A K M A T S S L R R M S Q L L S L R P D L R F T V A A I P P R N D P R D A V V - - - T R D E L V L A E L P A G S L V G T S S L R R A A Q L R A L G L G L

G F E L G G I T K R ~ D P T D C L ~ W P F Y ~ A Y K ~ L ~ D L P ~ G G I ~ G ~ ~ ~ ~ R R ~ A Q L K R K Y P H L

1451 1501 1551 1601 1651 1701 1751 180( La51 tqq i I r E L r g i i v g t r L s k L d n g e Y d A I I l a V A g L h r l g l e s ~ i r a a l p p i i I I R - S L H G N V G T H L H K L D F - - G E ~ D A I I L A V A G L K L G L E S R I - - - - H A A L P P E I E F R - S I R G N L N T R L R K L D E - Q Q E F S A I I L A T A G L Q R M G H H N R V - - - - G Q I L H P E K

E P K - S I R G N L N T R L R K L D E - L Q E F S A I V L A V A G L Q R ~ G W Q N R V - - - - G Q I L H P E E T V Q D N F R G N V Q T R L R K L S E - - G V V K A T L L A L A G L K R L N M T E N V - - - - T S T L S I D D

K F L - E L R G N V N T R L A K L D S - - G D Y D A I I L A A A G L K R L G F S D R V L P G E T N I I D P N V T I K - U I R G N I D T R L Q K L E T - - E D Y D A I I L A A A G L S R M G U K Q D V V - - - T E F L E P E R K F E - S V R G N I Q T R L Q K L D D P K S P Y Q C I I L A S A G L M R M G L E N R I - - - - T Q R F H S D T Q I R - F L R G N V N T R L A K L D R - - G E Y D A I I L A A A G V I R L G F E S R I - - - - R S S I S V D D K T A P - I R G N V Q T R L K K L D S - - G E F S A I V L A A A G I K R L G L E S R I - - - - G R Y F S V D E E I M D - I R G N L N T R F K K F D E - - G D F D A M M L A Y A G V Y R L E F S D R I - - - - T E I L - P H E E I R P - L R G N L D T R L N R V S S - - G D L D A I V V A R A G L A R P G R L D E V - - - - T E T L D P V Q

E F K - S I R G N L N T R L R K L D E - Q L E F ~ A I I L A V A G L Q R ~ G ~ Q N R V - - - - G Q I L H P E E

H V E E N F R G N V Q T R L S K L Q G - - G K V Q ~ ~ L L R L A G L K R L S ~ ~ E N V - - - - A S I L S L D E

1951 2001 2051 210/ 215) 2001 2251 2301 2351 2401 2451

S L - P A V G Q G A V G I E C R L D D S R T H E L L A A L ~ H H E T A L R V ~ A E R A M N ~ R L E G G ~ Q V P C M Y - A V G Q G R L G V E V R A K D Q D I L D L V G V L H D P E T L L R C I A E R A F L R H L E G G C S V P C M Y - A V G Q G A L A V E V R A K D Q D I L D L V G V L H D P E T L L R C I A E R D F L R H L E G G C S V P C M Y - A V G Q G A L A V E V R A K D Q D I L D L V S V L ~ D P E T L L R C I A E R A F L R H L E G G C S V P M L - P R V A Q G A I G I R C R S N D D K M A E Y L A S L N H E E T R L A I S C E R A F L T T L D G S C R T P M L - P A V A Q G A I G I A C R T D D D K M A T Y L A S L N H E E T R L A I S C E R A F L E T L D G S C R T P M C - P A A G Q G A L S I E L R T N D P E I A A L L E P L H H I P D A V T V A C E R A M N R R L N G G C Q V P C L - P A V G Q G A L A I E C R E S D E E L L A L F S Q F T D E Y T K R T V L A E R A F L N A M E G G C Q V P M Y - H A V G Q G A L G I E I R K G D T K M M K I L D E I C D L N A T I C C L S E R A L M R T L E G G C S V P S L - P R G G Q G A V G I E C R T A D S D L H A L L E P L H H T D T A L R V T A E R A L N K R L N G G C Q V P I L - P A A S Q G I I A V Q G R V G E N - - F D F L K L F H S E E S L C I S L A E R T F V R E M N G G C S T P T M L P A V G Q G A L G I E T R T D D A E T R E I V R V L N D D N T E M C C R A E E A L L R R L Q G G C S A P - M V P A P A Q G A I A V E C R A G D S R L V A V L A A L D D A D T R A A V T A E R V L L A E L E A G C S A P

~ ~ - ~ a v ~ ~ ~ ~ ~ ~ ~ ~ ~ i l d ~ ~ r ~ r e l ~ a a ~ n ~ h e ~ a i ~ ~ t ~ ~ r ~ ~ ~ t t ~ e ~ g ~ ~ v ~

(mntmuedi


TABLE V. Alignment of Known Amino Acid Sequences of PBGD With JOY'l E. coli Structural Environment Description on Uppermost Line of Alignment (Malign")* (continued)

E eoli Human Rat M O W

Pea hahidopsis Euglena Bacillus Yeast Pseudomonas C.JOSLIi C. utbmforrn M . kpme

2201 2551 2601 2651 2701 2751 I G S Y A ' ~ ~ ~ a q e ~ i ~ ? ~ ~ v ~ a p Z ~ B q ~ i ? q e ? r q a I G S Y R E L I - - D G E I W L R R L V G A P D G S Q I I R G E ~ R G A - - - - - - - - - - - - - - - - - - - V A V H T A M K - - D G Q L Y L T G G V W S L D G S D S I Q E T M Q A T I H V P A Q H E D G P E D D P Q L V G V A V H T V M K - - D G Q L Y L T G G V W S L D G S D S M Q E T f l Q A T I Q V P V Q Q E D G P E D D P Q L V G V A V H T V I K - - D G Q L Y L T G G V W S L D G S D S M Q E T M Q A T I Q V P V Q Q E D G P E D D P Q L V G I A G Y A S R D - K D G N C L F R G L V A S P D G T R V L E T S R I G S Y ~ - - - - - - - - - - - - - - - - - I A G Y R S K D - E E G N C I F R G L V A S P D G T K V L E T S R K G P Y V - - - - - - - - - - - - - - - - - I S G F A Q L K - - D G Q L R M E A R V G S V T G K G P L I I Q S K T F R L P W S G R T W P Q L - - - - - - - I A G Y S V L N - G Q D E I E M T G L V A S P D G K I I F K E T V T - - - - - - - - - - - - - - - - - - - - - I G V E S K P N E E T K K L L L K A I V V D V E G T E A V E D E I E M L I E - - - - - - - - - - - - - - - - - I A C Y A I R E - - G D Q L W L R G L V G Q P D G T Q L L R A E G R - - - - - - - - - - - - - - - - - - - - -

V G S F G S Y I - - - G T L K L L A F V G S V D G K - - - - - - - - - - - - - - - - - - T G L R N E V T K - - V G A I A Q V V - - - E S I D G E G R V F E E L S - - - L R G C V - - - - - - - - - - - A A L D G S D V I - -

I R A Y A T I Q G - S E I I L K G L Y C N - - - - - - - - - - - - - - - - - - - - - - - - - - E T T G E L - -

2801 2851 3 0 ) 295) 3001 305) 310) p q d A e q i G i s L A i ' e L l n n y A r e l L a e v y

E coh - - - - _ _ - - _ - P Q D A E Q M G I S L A E E L L N N G R R E I L A E V Y N G D A P A - -

Rat I T A R N I P R G A Q L A A E N L G I S L A S L L L N K G A K N I L D V A R Q L N D V R - - Mouse I T A R N I P R G A Q L A A E N L G I S L A S L L L N K G A K N I L D V A R Q L N D V R - - PeS - - _ _ - - _ - _ - Y E D M M K I G K D A G E E L L S R A G P G F F N S - - - - - - - - - - hahidopsis - _ _ _ - - - - _ - Y E D f l V K f l G K D A G Q E L L S R A G P G F F G N - - - - - - - - - - Euglena - - - - - _ - - _ - Q K E S E R L G V E V R D M L L A D G A Q A Y L D E A Y A S R T L G W A

Yeast - - - - - - - - N V K E D S M A C G K I L B E R M I R D G A K K I L D E I N L D R I K - - - Pseudomom - - - - - A P L A D A E A L G V R V A E D L L E Q G A E A I L E A V Y G E A G H P - - c psur - - R K E C V S G N R N N P V E L G Y E L V K K M - - K S S K S I C vrbroform - _ A V K T P E - - - - E A E A V G I E L A E I L L S M G A E K I L A D I R K T C M lepme - - - R A S G I S T S G R A S E L G L A V R V E L F E L G A R E L M W G A R S D R A R G S

Human I T R R N I P R G P Q L A A Q N L G I S L A N L L L S K G A K N I L D V ~ R Q L N D A H - -

Ba all us _ - _ - - - _ - _ _ G N D P E E V G K R C A A L M A D K G R K D L I D R V K R E L D E D G K

*JOY structural environment description key: X = solvent inaccessible (upper case), x = solvent accessible (lower case), = H-bond to other side chain (ti1de)x = H-bond to main chain amide (bold), x = H-bond to main chain carbonyl (underline), *=mobile loop.

the active-site cleft, as described above (see Figs. 10a and 11). The reduced dipyrromethane in the reduced native and selenomethionine variant occupies the C l and C2 sites. In the oxidized conformer of the native enzyme, however, ring C1 remains in the C1 site but ring C2 occupies a site (termed the S site) which may closely approximate to the site of substrate binding and polypyrrole-chain elongation. A porphobilinogen molecule bound at the S site would be appropriately situated to react with the free a-PO- sition of the terminal ring of the polypyrrole chain, which is positioned deeper in the active-site cleft. Thus in the reaction that converts E to ES1, this terminal ring is located at the C2 site. The substrate could conceivably bind in a reverse orientation (see Fig. 14), such that the positions of the acetate and propionate groups are interchanged. The two reacting atoms (the carbon at the free a-position of the terminal ring and the amino methyl group of the substrate, or its equivalent) would then be placed in closer proximity. The terminal ring of the polypyrrole chain (presumably when occupying the elongation or S site) is susceptible to cleavage and hydrolytic release as hydroxyporphobilinogen, particularly in the absence of exogenous porphobilinogen when the S site is free. The conformation adopted by the cofactor that directs the C2 ring away from the substrate-binding or elongation site may explain the stability of the dipyrromethane in PBGD against catalytic turnover.

The proposed roles of the identified pyrrole bind-

ing sites correlate well with results from mapping of the active site through arginine mutagene~ i s . ' ~ '~~ Thus Argl31 and Arg132 form key interactions at the C1 site, since replacement of either completely prevents dipyrromethane cofactor assembly and confirms the importance of these amino acids. Argll, Arg149 and Arg155 form interactions at the proposed substrate-binding site (S site) but Arg155 has a dual role, binding at the C 1 site as well as the S site. Capacity to assemble the cofactor is retained by mutations at these residues. However, replacements of Argll and Arg155 severely inhibit conversion of E to ES,. An Arg149 mutant has a greatly elevated K, and is inhibited at all stages of product elongation, particularly the ES, to ES, step.

The interactions between the substrate porphobilinogen and the protein at the S site are formed mainly through the acetate and propionate side-substituents (Fig. 14). This explains why analogues of porphobilinogen, in which these substituents have been esterified or replaced with alkyl groups," act as poor substrates. The substrate-binding site S has suf- ficient space to accommodate only a single porphobilinogen moiety, which would explain the observation that PBGD cannot readily incorporate di- or tripyrromethanes into the 1-hydroxymethylbilane product.,

The porphobilinogen molecule would be expected to carry a net negative charge at neutral pH. Thus, the excess of positively charged amino-acid side- chains (including Argll, Lys83, Argl31, Arg132,

STRUCTURE OF E . COLZ PBGD

TABLE VI. Invariant Residues in PBGD

71

Residue Structural environment and possible roles of side chain

Argl l Serl3 Leu15 Ala16

Gln19 Gly45 Asp46 Leu53 Gly57 Lys59 Leu61 Lys64 Glu65 Leu70 Asp76

Val79 His80 Ser81 Lys83 Asp84 Pro86 Pro90 ArglOl

Asp106

Leu116 Gly126 Thr127 Arg132 Gln135

Pro141 Leu143 Arg149 Gly150

Asnl51 Thr154 Arg155 Ala170 Gly173 Arg176

Ala195 Gln198

Gly199

Arg206 Glu231

Arg232

cys242 Pro245

Glv287

Forms salt bridge with propionate of cofactor ring C2 and with Asp46 from lid region. Hydrogen bonds propionate of substrate; forms N-cap for helix all. Lines the rear of active-site cleft. Directed into interior, adjacent to aliphatic portion of Argl l side chain; Requirement for small

Lines active-site cleft and forms hydrogen bonds to side chains of conserved Ser81 and Arg176. Located adjacent to Phe62 at interface between two portions of lid regi0n;restricted steric space. Forms salt bridge to Argl l guanidinium group, linking lid region with body of molecule. [Occur in- -flexible- -lid region- -of molecule]. Forms intrahelical salt bridge to Glu67 side chain. Bridges the amino ends of two a-helices (a2a1 and a2,). Packed in hydrophobic core. Forms salt bridges to guanidinium groups of both Arg7 and Arg206; may have role in structural

Packed in hydrophobic core. Lines floor of active-site cleft; forms salt bridge to Glu204 side-chain carboxylate. Located below cofactor, hydrogen bonds propionate of cofactor ring C2 and Gln19 carboxamide. Forms salt bridges to cofactor (acetates of both ring C1 and ring C2. Hydrogen bonds the two pyrrole nitrogens of cofactor; role in catalysis. Terminates short 3,,-helical segment; role in protein folding. Surface position; first residue in @-turn. Involved in interdomain interactions: forms buried salt bridge to Glu231 and hydrogen bonds 198 0;

Forms salt bridges to both guanidinium groups of both Argl31 and Arg132; stabilizes

Packed in hydrophobic core. Steric requirement for small side chain. Hydrogen bonds propionate of cofactor ring C1. Forms salt bridge with propionate of cofactor ring C1. Semi-buried position, forms hydrogen bonds to buried polar groups OEl - 193 N and NE2; 131 0 and

Terminates helix al, and is second residue of turn; role in protein folding. Packed in hydrophobic core. Forms salt bridge with acetate of substrate. Adopts otherwise disallowed conformation ( + 128"); amide-nitrogen hydrogen bonds acetate of cofactor

Hydrogen bonds Argl l guanidinium group in domain 2-1 interaction. Located on surface. Forms salt bridge with propionate of cofactor ring C l and with acetate of ring C2. [Located along one face of a3,-helix, forming rear wall of Ala172 active-site cleft, small- -side-chains may be necessary to provide volume to accommodate the growing polypyrrole chain.] Located at bottom rear of active-site cleft, guanidinium group interacts with propionate of cofactor

interactions with growing polypyrrole chain. Lines right hand rear of active-site cleft. Forms hydrogen bonds to side chains of AsplO6, Lys83, and Argl31, and 104 0; stabilizes

Third residue of type-I1 @-turn within second hinge-strand, located in position of limited steric space

Forms salt bridges with Asp76, Asp209 and hydrogen bonds to 70 0 and 75 0; stabilizes protein fold. Forms interdomain, buried salt bridge to ArglOl, and hydrogen bonds to backbone amide-nitrogens

Guanidinium group hydrogen bonds backbone carbonyl-oxygens, 85 0 and 86 0; forms stabilising

Covalently binds cofactor. Packed within predominantly hydrophobic cluster of side chains; may confer specific conformation to

cofactor binding loop.

aliphatic side chain.

stabilisation.

stabilizes protein structure.

hydrogen-bonding network surrounding cofactor.

193 0; stabilizes protein fold.

ring C2.

ring C2 via a bridging water molecule, and with Gln19 side chain and 166 0. May form

hydrogen-bonding network surrounding the cofactor .

at bottom rear of the active-site cleft.

197 N and 250 N; stabilizes protein fold.

interdomain interactions.

Located a t sterically restricted site at interface between two a-helices; stabilizes protein fold.


Fig. 14. Interactions formed by a porphobilinogen molecule at the proposed substrate binding site. The position of the substrate porphobilinogen molecule has been modeled on the observed position of the C2 ring in the oxidized form of the dipyrrole. Similar hydrogen-bond and salt-bridge interactions (drawn in dotted lines) between the substrate (dark gray) and polypeptide-chain groups (mid-gray) are possible with two distinct orientations of the por-

phobilinogen molecule, which differ mainly in the positioning of the side-substituents. In (b), the position of the acetate and propionate groups are interchanged from those of the C2 ring in the oxidized dipyrrole (a), and thus the aminomethyl group of the porphobilinogen group is closer to the nucleophilic free a-carbon of the dipyrromethane cofactor (light gray).

Arg149, Arg155, and Arg176) within the active-site cleft may also play a role in the initial attraction of porphobilinogen to the cleft. The binding of porphobilinogen may also be facilitated by the lack of acidic side-chains on the protein surface immediately surrounding the active-site cleft.

Mechanism of catalysis of the ring couplings The three-dimensional structure of the PBGD ac-

tive-site shows no indication of replicated catalytic machinery, thus ruling out the possibility that the series of ring couplings are catalyzed at (up to) four distinct sites within the active-site cleft. This con-

Nayak

between the substrate (dark gray) and polypeptide-chain groups (mid-gray) are possible with two distinct orientations of the por-


clusion is consistent with biochemical evidence for the presence of only a single catalytic site.’ Asp84 is identified as the key catalytic residue, owing to the close proximity of its carboxylic-acid group to the pyrrole nitrogens of the cofactor and the greatly di- minished activity of mutant proteins in which Asp84 is repla~ed.~’ Thus the mutant Glu84 retains less than 1% of the specific activity of the wild-type protein, whereas the Ala84 and Asn84 mutants have no catalytic activity. Other polar residues in the active-site cleft serve mainly to bind the porphobilinogen substrate and the growing polypyrrole product.

A model can be proposed for the role played by Asp84 in the sequence of reactions involved in ring coupling. The side-chain carboxylic-acid group may initially be protonated, as it is inaccessible to the external solvent medium and is positioned approximately normal to the face of the adjacent Phe62 phe- nyl ring (see Fig. lla(ii)). The two oxygen atoms of the carboxylic acid can interact with the hydrogen atoms attached to both pyrrole and primary-amino nitrogens of the porphobilinogen substrate to facili- tate its deamination: one oxygen atom can provide the proton to be taken up by the expelled ammonia molecule (to yield an ammonium ion) and the result- ing carboxylate anion can stabilize the carbocation that may develop at the exocyclic carbon atom; the other oxygen can stabilize the positive charge that developson the pyrrole nitrogenoftheresultingmeth- ylene pyrrolenine. It has been suggested that the acetate side-substituent of the substrate molecule has a role in intramolecular stabilization of the car- b ~ c a t i o n , ~ ~ although this would seem improbable as the acetate is occupied in forming salt-bridges with several arginine side-chains (Argll, Arg149, and Arg155). Following the deamination, the free oxygen of the Asp84 carboxylate group can stabilize a positive charge on the pyrrole nitrogen of the terminal ring of the polypyrrole chain, thus promoting the generation of a nucleophilic carbon atom at the free a-position of this ring. After carbon-carbon bond formation, the carboxylate group of Asp84 can remove the proton from the a-position of the now penultimate ring to complete the ring-coupling process.

To initiate the final step in preuroporphyrinogen synthesis, the Asp84 carboxylic acid protonates the carbon at the a-position of ring C2 of the dipyrromethane cofactor in ES4.72 Next, the carboxylate group promotes the reversal of a ring-coupling reaction to release a tetrapyrrolic methylene pyrrolenine. Finally, the nucleophilic attack of a water molecule to the exocyclic carbon-carbon double bond in the methylene pyrrolenine completes product formation. A water molecule (Wat416) (see Fig. l l a / b(ii)), hydrogen-bonded to the Asp84 side-chain, may be the origin of the hydroxyl group in the final 1-hydroxymethylbilane product, preuroporphyrinogen.

The proposed model ostensibly suggests that Asp84 alone can promote the catalysis of all the reaction steps involved in ring coupling. However, the importance of the binding interactions is underlined by kinetic data on site-directed mutants.72 For ex- ample, preliminary results indicate that the Asp84Ala and Asp84Asn variants, although inactive in forming product, retain the capacity to assemble the dipyrromethane cofactor and may also couple one or two molecules of substrate in what are not likely to be true catalyzed events. As discussed above, amino-acid replacements of Argl31 and Arg132 at the C1 binding site completely prevent cofactor assembly and result in an inactive apoenzyme. However, mutation of Lys83, that forms salt- bridges with both rings C 1 and C2 and stabilizes the cofactor conformation so that the ring C2 is directed toward the rear of the active-site cleft, results in a protein unable to fold (unpublished observation). Thus binding of the two reacting pyrrole rings in the appropriate relative positions must play the major part in promoting ring coupling. In addition, upon binding of the substrate to PBGD, a stabilizing intramolecular ion-pair between the acetate and aminomethyl groups of the p~rphobilinogen~~ would be broken, further promoting deamination of the incoming porphobilinogen.

As described above, the exocyclic methylene carbon of the deaminated substrate porphobilinogen and the pyrrole nitrogens of the terminal ring of the polypyrrole chain (as well as that of the substrate molecule) may transiently acquire positive charges during the ring-coupling reactions. This may provide an explanation for the absence of an a-helix in the segment of polypeptide chain corresponding to 013,. The amino-end of an a-helix here would point directly a t the elongation site, thus disfavoring these positive charges.

Flexibility around the active-site clefi The presence of only a single catalytic site in the

PBGD molecule requires that the growing polypyrrole chain must be repositioned after each ring-coupling step to allow the a-carbon of the terminal pyrrole ring to be aligned for further reaction in the tetramerization. Considerable flexibility in the vicinity of the active site is required: (1) to permit entry of the substrate, (2) to allow conformational adjustments associated with both attachment of the cofactor and extension of the polypyrrole, and (3) the release of the hydroxymethylbilane product. This flexibility is suggested by several structural features of the PBGD molecule.

First, the loop of polypeptide chain that carries the cofactor makes few interactions with the rest of the molecule and has relatively high temperature factors (see Figs. 8 and 12). Within this loop, Cys242 is preceded by two glycine residues that would allow considerable flexibility of the cofactor loop.


Second, the main interactions between the protein and the porphobilinogen moieties of the cofactor (and presumably the substrate) are made through the acetate and propionate side-substituents. Conforma- tional adaptability in these extended side-groups could enable favorable protein-polypyrrole interactions to be maintained throughout the course of product elongation. This adaptability is apparent in the similar positioning of the ring C 1 carboxylate groups in the reduced and oxidized conformers of the cofactor dipyrrole, despite the somewhat differing positions of the pyrrole rings themselves (see Fig. 10).

Third, the loop of polypeptide chain comprising residues 48-58, that is thought to be located directly in front of the active-site cleft, must be highly mobile at pH 5 since the electron density is poorly resolved and may point to the presence of a flexible “lid.” This “lid” may function: (1) to form additional binding interactions with the substrate, and (2) to seal off transiently the active site during catalysis. This may be important both for protecting reactive intermediates from exposure to solvent and for modulating the nature of the active-site environment. The proposed role for this segment of polypeptide chain is consistent with several observations. For instance, lowered enzymic activity results from replacement of Lys59 by glutamine or chemical modification by pyridoxal phosphate of both Lys55 and Lys59, which occur within the Furthermore, the presence of the substrate, porphobilinogen, affords these lysines pro- tection from the pyrid~xylat ion.~~ The ordered polypeptide chain on either side of the “lid” segment is more poorly defined in the reduced form of the deaminase, in which the substrate-binding site is vacant,

than in the oxidized protein, in which the S-site is occupied by the ring C2 of the oxidized dipyrrole system. Interactions that may be responsible for or- dering the “lid” region upon substrate binding include the salt-bridge between Asp46 and Argll (Argll in turn salt-bridges the propionate of the porphobilinogen moiety) and the stacking of Phe62 against the pyrrole ring (see Fig. 11).

Fourth, the location of the active site at the interface between the three domains must maximize the effect of the interdomain flexibility which could be of importance in view of the following factors:

i) The cavity between domains 1 and 2 has a large volume capable of holding several porphobilinogen- derived pyrrole units (as discussed below). ii) There are few direct interactions between the domains 1 and 2. iii) Domains 1 and 2 potentially can hinge or twist open, and thus increase the volume of the interdomain cleft and change the relative position of the residues lining the cleft that are contributed by each of the domains. iv) Such interdomain hinge-opening has been clearly demonstrated in the structurally analogous binding-proteins such as lact~ferr in?~ maltodextrin-binding protein,76 and lysinelargininelorni- thine-binding protein77 (see also ref. 78, and references therein). v) Domain 3 interacts with the rest of the molecule primarily through hydrogen-bonds and salt-bridges. Thus upon movement of domain 3 away from the other two domains, the exposure of largely hydro- phillic surfaces to solvent would be favorable.

Fig. 15. Internal cavity in the active-site cleft of wild-type PBGD. The surface of the cavity is outlined by the dot surface calculated by the method of Conn01ly~~ with a probe radius of 1.4 A. Residues lining the cavity are labeled. The orientation of view is from the rear left relative to that in Figure 9a.

STRUCTURE OF E . COLZ PBGD 75

A-

- Fig. 16. Location of invariant residues in the PBGDs. The backbone of E. coli PBGD is drawn

as an @-carbon skeleton, on which 56 of the invariant residues are shown. Two invariant residues, Leu53 and Gly57, occur in the unobserved flexible loop and are therefore not shown.

There is considerable biochemical evidence for a change in the relative positioning of domains in the PBGD molecule over the course of the complete reaction cycle from protein modification studies with N-ethylmaleimide. The susceptibility to modification of cysteine-134 by this reagent increases pro- gressively as PBGD proceeds from the holoenzyme (E) through ES,, ES,, to ES,.79 Cys134 is located at the interface between domains 2 and 3 (see Fig. 8) suggesting that its environment changes substan- tially during the catalytic cycle. Furthermore, modification of this sulphydryl group, which is -18 A distant from the catalytic site (Asp84), inactivates the enzyme and destabilizes the protein. In addition, substitution of Arg232 for histidine also inhibits conversion of ES to ES,.l6 Arg232 is an invariant residue that is also remote from the active site (-10 A from Asp84) but forms hydrogen-bonds linking domain 3 to domain 1. Finally, the anomalously large difference in the chromatographic mobilities of ES, and ES, suggests a major conformational change occurs upon incorporation of the third pyrrole ring.

Manipulation of the growing polypyrrole chain

A possible model for the manipulation of the growing polypyrrole chain is for the chain to fold into the large active-site cavity (Fig. 15) displacing bound water molecules. Interestingly, the calculated vol-

ume of the cavity in the protein is large enough to accommodate a polypyrrole chain of approximately 3 112 porphobilinogen units in length. Thus, this model provides an obvious steric mechanism for re- stricting chain growth beyond a specific length (i.e., a tetrapyrrole). This model also provides an explanation for two observations: (1) the intriguing conservation of residues lining the active site cavity, in particular the small side-chains Ala170, Ala172, and Gly173 which line the inward face of the a3,- helix; (2) the inhibited formation of ES, from ES, upon replacement of Arg176, which is located at the rear left of the cleft (as shown in Figs. 15 and 16). An important aspect of this model is that the catalytic and substrate-binding sites are formed by residues primarily from domain 1, whereas the binding sites for the cofactor are formed by residues primarily from domain 2. Thus, as the polypyrrole chain grows within the active-site cleft forming interactions mainly with domain 2, shifts in the position of domain 1 can carry the catalytic site and bound substrate into the appropriate position to react with the terminal pyrrole ring of the chain. An appropriate name for this model would be the sliding active site model.

Other models are also possible. One, termed the moving chain model requires that the polypyrrole chain is pulled processively past the catalytic site, through rigid-body movement of all, or part, of domain 3 (to which the cofactor and growing chain is


anchored). In this model, the newly extended polypyrrole chain would need to be repositioned, after every substrate addition, so that the binding sites C1 and C2 again hold the penultimate and terminal rings, respectively, to allow the preceding ring($ to be disdaced from the active-site cleft. Thus. with

G.W.J. Chemical and enzymic studies on biosynthesis of the natural porphyrin macrocycle: Formation and role of unrearranged hydroxymethylbilane and order of assembly of the pyrrole rings. Bioorganic Chem. 8:451-463, 1979.

7. Seehra, J.S., Jordan, P.M. Mechanism of action of PBGD: Ordered addition of the four porphobilinogen molecules on the formation of preuroporphyrinogen. J. Am. Chem. SOC. 102:6841-6844. 1980.

each ihain extension, the binding interactions at both the C1 and C2 sites are broken and subse- quently reformed with the succeeding porphobilinogen units of the growing polypyrrole. It would seem, however, that the breakage of the numerous, highly specific interactions occurring at each binding site may present a relatively high energy barrier for each reaction cycle in such a model.

We are actively engaged in further structural studies of mutants and enzyme-intermediate complexes to investigate the mechanism further. The structures of R149H and R176H mutants a t medium resolution have confirmed that no gross conformational changes accompany the loss of activity.” Me- dium resolution studies of the Asp84Glu mutant, however, suggest that the considerable loss of activity associated with this amino acid replacement may be related to the loss of a hydrogen-bond between the side-chain and the pyrrole nitrogen of cofactor ring C2. Work is in progress to refine this structure at high resolution with data collected at 100°K where the “lid” may be more clearly defined.

ACKNOWLEDGMENTS We thank the Biotechnology and Biological Sci-

ences Research Council (UK) and the Swiss Na- tional Science Foundation for financial support of this work, and the Rayne and Wolfson Foundations for refurbished laboratories a t Birkbeck College. We thank A.R. Battersby, J.N. Jansonius, F. Leeper, and A. Pitt for helpful discussions, R. Pickersgill for assistance in data collection on the uranyl sulphate derivative, and N. Srinivasan for assistance with Figure 14. G.V.L. was an MRC Canada supported research fellow.

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Documents

The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-Å resolution