Structural and Sequence Comparisons of Quinone Oxidoreductase, ζ-Crystallin, and Glucose and Alcohol Dehydrogenases

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 328, No. 1, April 1, pp. 173–183, 1996Article No. 0158

Structural and Sequence Comparisons of QuinoneOxidoreductase, z-Crystallin, and Glucose andAlcohol Dehydrogenases

Karen J. Edwards,*,1 John D. Barton,* Jamie Rossjohn,† Jennifer M. Thorn,* Garry L. Taylor,‡ andDavid L. Ollis**Centre for Molecular Structure and Function, Research School of Chemistry, Australian National University, Canberra,Australian Capital Territory 0200, Australia; †St. Vincent’s Institute of Medical Research, Fitzroy, Victoria 3065,Australia; and ‡School of Biology and Biochemistry, Bath University, Claverton Down, Bath BA2 7AY, United Kingdom

Received September 8, 1995, and in revised form January 15, 1996

single protein fold can be used for a number of func-Quinone oxidoreductase, z-crystallin, glucose dehy- tions, the aspects of a structure that make it suitable

drogenase, and alcohol dehydrogenase belong to a for multiple functions are not obvious. In the casesuperfamily of medium-chain dehydrogenase/reduc- of enzymes the situation is more complex: they aretases. The crystal structures of Escherichia coli qui- capable of binding substrates and of catalyzing reac-none oxidoreductase (QOR) and Thermoplasma acido- tions between bound groups. In this paper, attentionphilum glucose dehydrogenase have recently been de- is focused on two new enzyme structures which aretermined and are compared here with the well-known structurally homologous to horse liver alcohol dehy-structure of horse liver alcohol dehydrogenase. A drogenase (LADH).2 All three are redox enzymes andstructurally based comparison of these three enzymes share a common three-dimensional structural foldconfirms that they possess extensive overall structural yet exhibit dissimilar cellular functions and sub-homology despite low sequence identity. The most sig- strate specificities. The most obvious differences arenificant difference is the absence of the catalytic and associated with the zinc ions that are bound to somestructural zinc ions in QOR. A multiple structure-

of these proteins and with their catalytic residues.based sequence alignment has been constructed forIn LADH these bound metals are responsible for thethe three enzymes and extended to include z-crys-catalytic activity and structural stability of the pro-tallin, an eye lens structural protein with quinone oxi-tein. Despite the importance of zinc ions in LADH,doreductase activity and high sequence identity to E.other members of the family function effectively incoli quinone oxidoreductase. Residues which are im-the absence of bound metal ions.portant for catalysis have been altered and the func-

Alcohol dehydrogenases occur in a wide range of or-tions and activities of the enzymes have diverged, il-ganisms spanning the Eukarya, Bacteria, and Archaealustrating a classic example of divergent evolution(1–3). A medium-chain dehydrogenase/reductaseamong a superfamily of enzymes. q 1996 Academic Press, Inc.

(MDR) family has recently been defined, extended, andKey Words: alcohol dehydrogenase; glucose dehydro-genase; medium chain dehydrogenase; quinone oxido- subdivided to include sequence-related dehydroge-reductase; z-crystallin. nases and reductases (4). Those classified as dehydro-

genases require zinc ions (the Zn-ADHs) for activity,whereas the reductases do not necessarily requiremetal ions for activity. The Zn-ADHs catalyze the re-

Despite the rapid rate at which new protein struc-tures are being determined, the appearance of a new 2 Abbreviations used: QOR, quinone oxidoreductase; LADH, liverprotein fold remains a rarity. While it is clear that a alcohol dehydrogenase; GDH, glucose dehydrogenase; Zn-ADH, zinc

alcohol dehydrogenase; MDR, medium-chain dehydrogenase/reduc-tase; NADH, nicotinamide adenine dinucleotide; NADPH, nicotin-

1 To whom correspondence should be addressed. Fax: 61-6-249 amide-adenine dinucleotide phosphate; DMSO, dimethyl sulfoxide;rms, root mean square.0750.

1730003-9861/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

/ 6b15$$9366 02-27-96 12:06:43 arcas AP: Archives

174 EDWARDS ET AL.

FIG. 1. Pairwise superposition of the Ca backbones for (a) QOR and GDH and (b) QOR and LADH monomers. QOR is shown in black ineach figure; GDH and LADH are shown in gray.

duction of a wide variety of medium- to long-chain alco- The X-ray crystal structures of two further represen-tative members of this MDR superfamily have recentlyhols, with the horse LADH being the most extensively

studied (5, 6). LADH is homodimeric, with each mono- been reported. These are an Escherichia coli quinoneoxidoreductase (8, 9), which catalyzes the NAD(P)H-mer consisting of a catalytic and nucleotide-binding do-

main. The catalytic domain comprises mainly b-sheet dependent reduction of quinone substrates, and a Ther-moplasma acidophilum glucose dehydrogenase (10),structures while the nucleotide-binding domain con-

tains the characteristic babab motif known as the which catalyzes the conversion of glucose to gluconate.These three enzymes have several functional and struc-‘‘Rossman fold’’ (7). LADH specifically requires the co-

factor NAD/ for activity. tural differences. GDH belongs to the zinc-containing


175COMPARISON OF FOUR MEDIUM-CHAIN DEHYDROGENASES

TABLE I

Pairwise Ca rms Deviations between QOR:LADH and QOR:GDH.

Monomer Dimer

Domains aligned: LADH GDH LADH GDHa

Catalytic domain 1.9 A (158) 3.6 A (149) 2.7 A 7.6 ADinucleotide binding domain 2.5 A (139) 3.5 A (127) 3.7 A 4.7 AAll common Ca 2.7 A (297) 3.8 A (261) 3.7 A 7.5 A

Note. The number of atoms used in the calculation is given in parentheses.a One-half of the GDH tetramer, composed of two monomers equivalent to the QOR and LADH dimers.

art, unpublished results) and used to construct a structure-baseddehydrogenase subfamily, as does LADH, whereassequence alignment within the GCG package (15). Since QOR is inQOR is a member of the reductase subfamily and doesits binary complex with the reduced form of NADP, comparisons

not possess metal atoms. In addition, both QOR and were made to a similar LADH complex. Coordinates for the LADH/LADH are dimeric, whereas GDH is tetrameric. De- NADH/DMSO crystal structure were obtained from the Brookhaven

Databank (16) [Accession No. PDB2OHX.ENT (17)]. Coordinates forspite the low sequence identity between these threeQOR are available from the Brookhaven Databank (Accession No.enzymes (LADH and GDH have 21 and 17% sequencePDB1QOR.ENT) and those for GDH may be obtained from Garryidentity with QOR, respectively), they exhibit exten-Taylor. z-Crystallin (guinea pig) was included in the alignment due

sive structural homology, not only in their nucleotide- to its significant sequence identity and activity similar to that ofbinding domains but also in their catalytic domains. QOR. On the basis of the structure-based sequence alignment, pair-

wise least-squares fitting of coordinates was carried out for commonThe MDR superfamily contains a number of otherCa atoms in QOR and LADH and QOR and GDH. Visual inspectionenzymes which have been identified on the basis ofof these alignments gave refined common structural regions whichtheir sequence similarity. These have been detailed by were used for final pairwise alignments and rms deviation calcula-

Persson and coworkers (4). Of particular importance to tions. Only those residues which were clearly homologous were usedthe present study is the family of z-crystallins which, for the final alignments.like QOR, belong to the reductase subfamily of MDRs. Accessible and electrostatic surfaces. Accessible surface areas

were calculated with X-PLOR (18) using the algorithm of Lee andThe z-crystallins also have quinone oxidoreductase ac-Richards (19) with a probe radius of 1.6 A. Solvent-accessible surfacestivity and require NADPH as a cofactor (11). z-Crys-were displayed with the Alberta/Caltech version of FRODO/TOMtallin is a major structural eye lens protein of camels, (20) used in conjection with Connolly’s ‘‘ms’’ program (21). Electro-

llamas, and some hystricomorphic rodents, and ap- statics for the substrate-binding site were determined using GRASPpears to belong to a group of crystallins which have (22) (data not shown).been recruited from functional enzymes (12). Extensivesequence-based alignments and comparisons have RESULTS AND DISCUSSIONbeen performed to analyze structure–function and evo-

Figure 1 clearly shows that the overall structures oflutionary relationships between members of the MDRthe QOR, GDH, and LADH monomers are similar. Ta-superfamily (4, 13, 14). It should be noted that theseble I gives rms deviations between QOR, GDH, andstudies have been based exclusively on the structureLADH monomer subunits and dimers. Superpositionof LADH.of the structures of the three enzymes enabled a struc-Here we compare the structures of QOR, GDH, andturally based sequence alignment to be constructed andLADH to find the elements of their structure which arethe relationship between sequence and structural con-most important for their function and which have beenservation examined.conserved during the course of evolution. A structure-

based sequence alignment has been constructed for theStructure-Based Sequence Alignmentthree enzymes and extended to include z-crystallin.

QOR, GDH, and LADH have strong overall similarity A multiple structure-based sequence alignment haswith numerous structurally conserved regions. Func- been constructed for the three MDR enzymes LADH,tional considerations relating to both the nucleotide- GDH, and QOR. The sequence of z-crystallin was addedbinding and the substrate-binding sites are discussed. to this alignment due to its similarity to QOR. Figure

2 shows the alignment of QOR, GDH, LADH, and z-METHODS crystallin with boxed areas representing structurally

conserved regions. These regions predominantly con-Structurally based sequence alignment. The structures of QOR,GDH, and LADH were superposed using the program SHP (D. Stu- tain secondary structural elements. The largest differ-


176 EDWARDS ET AL.

FIG. 2. Structure-based sequence alignment of QOR (Q), LADH (L), GDH (G), and z-crystallin (Z). Residue numbers for each protein aregiven in parentheses at the end of each line and secondary structure elements are labeled for QOR. The catalytic domain of QOR encompassesresidues 2–124 and 266–327; the nucleotide binding domain contains residues 125–265. Residues identical in all four proteins are shadedin dark gray while similar residues are shaded in light gray. Residues involved in binding the catalytic zinc of GDH and LADH are marked(M). Residues which bind the structural zinc of both enzymes (F), GDH (FG), and LADH (FA) are also marked. Boxed areas representstructurally homologous regions of the three proteins.

ences are found mainly in the loop regions connecting Although the relationship between these enzymeswas predicted on the basis of their sequence alignmentssecondary structure elements where numerous inser-

tions and deletions are to be found. Despite the fact (4, 13, 14), the exact structural homology between theseenzymes would have been difficult to predict basedthat the sequences of QOR, GDH, and LADH possess

very few strictly conserved residues (21 in total, of solely on sequence comparisons. In the light of the pres-ent three-dimensional structural information the mul-which 9 are glycine) and low sequence similarity, it

can be seen that extensive structural homology exists tiple sequence alignment reported for these proteinsand the other MDRs (4) could be significantly im-between these enzymes. This is evident not only in

the nucleotide-binding domain but also in the catalytic proved. One of the reasons for poor sequence alignmentis that although most insertions and deletions occur indomain.



FIG. 3. Sequence alignment for QOR (Q), GDH (G), and LADH (L) extracted from that reported by Persson et al. (4), showing the correctedalignment based on the structures of the enzymes. Secondary structure elements for QOR are labeled. The lines connecting residuesrepresent the correct sequence alignment based on the structural alignment.

loop regions linking core secondary structures, errors of these enzymes. In particular, specific functions can-not be assigned to individual residues with any cer-are often attributable to misplacing of a gap in a core

secondary structure region. Choice of scoring schemes tainty. Additionally, phylogenetic trees are constructedfrom such multiple sequence-based alignments andand gap penalty functions for the alignment algorithm

may also affect the accuracy of the alignment. Factors may thus be misleading. Since any inferences from asequence alignment are dependent upon its accuracy,which may have affected the accuracy of the alignment

of the MDR enzymes (4) are the low sequence identity we would caution that care should be exercised whenmaking structure–function predictions based solely onbetween LADH and GDH and the use of a single struc-

tural model. sequence comparisons of evolutionarily divergent pro-teins with low sequence identity. The accuracy of se-Figure 3 shows the sequence alignment for QOR,

GDH, and LADH extracted from Persson and cowork- quence alignments can be improved by the inclusion ofadditional structural information into the gap penaltyers (4). As can be seen there are numerous regions

where there are discrepancies. The most significant is function (23) of the alignment algorithm. This can effec-tively be achieved by using more accurate secondarythe poor alignment of the structural Zn loop region for

GDH (residues 90–127). The region from aC to bF, structure information derived from a number of knownstructures within an enzyme family.which constitutes greater than half the Rossman fold

and includes the nucleotide-binding motif, is also Homology modeling can be a useful technique forpredicting the structure of an amino acid sequencepoorly aligned. This is of particular concern since the

Rossman fold is one of the most highly conserved pro- modeled from the known structure of a protein with ahomologous sequence (24). An initial step in homologytein folds known and many predictions are based upon

its structure. The second part of the catalytic domain modeling involves the construction of a three-dimen-sional framework for the structurally conserved re-(a4, b11, a5, and b12) is also misaligned. Such flaws

in the sequence-based alignments have the potential gions within a protein family (25). In many cases abetter model is produced from a framework rather thanto result in incorrect assumptions about the structure


178 EDWARDS ET AL.

GDH and z-crystallin are tetrameric. GDH tetramercontacts are predominantly via an additional loopfound between a2 and bA and the structural lobe. Asthis loop is absent in z-crystallin the tetrameric associ-ation of z-crystallin must be different from that ofGDH. The only residue involved in GDH tetramer for-mation which is conserved with z-crystallin (Gly208:GDH) is strictly conserved across all four enzymes.The major site of dimer–dimer interactions is strandbF. Although this region is structurally conserved,there is no corresponding sequence identity. The onlyconserved residue involved in dimer formation (Gly239:QOR) is in a region of minor interaction. As a re-sult, the detailed subunit interactions between theseenzymes differ markedly leading to significantly largerrms deviations for the dimer compared to the monomer(Table I).

The nucleotide-binding fold and mode of cofactorbinding appear to be structurally conserved throughoutthe dehydrogenase family (5, 26). The arrangement ofsecondary structural elements is highly conserved inthe nucleotide-binding domains of QOR, GDH, andLADH. The main difference between GDH, QOR, andLADH is that GDH is missing helix aD. This additionalhelix, found between the two bababmotifs of QOR andLADH, is not part of the classic Rossman fold (27),although it appears to be common in the MDRs (4).Secondary structure elements in the catalytic domainare also well conserved although the loop between helixa1 and strand b5 is quite different in all three enzymes.Strand b12 is absent in GDH, resulting in the forma-tion of a four stranded b-sheet compared to the six-

FIG. 4. MOLSCRIPT (32) diagram of the structure of QOR with stranded sheet present in QOR and LADH (Fig. 2).bound NADPH cofactor. The shaded areas represent the structurally The most significant difference between QOR andconserved regions found in QOR, GDH, and LADH and correspond to the other two enzymes is the absence of the catalyticthe boxed regions in Fig. 2. These are shaded as follows to represent

and structural zinc ions. Although the overall struc-conservation in each of the domains: light gray (catalytic domain),tures of the substrate-binding sites are similar, thedark gray (interconnecting a-helices), and midgray (nucleotide-bind-

ing domain). The area colored black corresponds to residues 87–94 equivalent catalytic zinc-binding residues in QOR have(QOR) and represents the region of the zinc-loop deletion in QOR. been replaced by residues which are incapable of coor-Secondary structure elements are labeled. dinating metal ions. The large structural zinc-binding

loop (Fig. 5) found in LADH and GDH is completelyabsent in QOR. The absence of this loop in QOR in-creases the accessibility of the substrate-binding cleftfrom an individual structure. On the basis of the struc-to solvent. Table II lists the solvent accessibility of theture-based sequence alignment of these enzymes, aactive site for all three enzymes and also gives the areastructural framework for the MDR enzymes is pro-covered by the structural zinc-binding loop in GDH andposed. QOR was chosen as the basis for this frameworkLADH. The diminished (in GDH) or absent (in QOR)as it is the simplest of the three representative en-structural zinc loop also exposes a large cleft whichzymes. This framework is depicted in Fig. 4 where theruns through the catalytic domains of GDH and QOR.structurally conserved regions are proposed to definePrevious workers (28) have pointed out that z-crys-a structural framework for the MDR enzymes.tallins do not have this structural zinc-binding loopnor do they have residues which are appropriate for

Overall Structure Comparison binding the catalytic zinc.The presence or absence of the zinc-binding loop alsoThe quaternary structures of the enzymes are some-

what different. LADH and QOR are dimeric, whereas affects the arrangement of the secondary structure at



FIG. 5. Zinc-binding loops and nearby regions. Ca traces are shown for QOR (thick gray), GDH (black), and LADH (thin gray). StructuralZn atoms for GDH (black) and LADH (gray) are also shown. The N- and C-terminal residues of the zinc-binding loops of GDH (residues90–127) and LADH (91–145), as well as the equivalent peptide of QOR (87–94), are marked (Zn N and Zn C). The ends of helix a3 of QOR(266–276) and LADH (319–325) and the equivalent peptide of GDH (305–308) are labeled. Helix a2 is labeled (QOR, 116–136; GDH, 149–167; LADH, 167–187).

some distance from the loop itself. From the superim- proteins. The degree and direction of this kink varies,resulting in the N-termini of helix a2 aligning well inposed structures (Fig. 5), it can be seen that the zinc

loops of LADH and GDH pass close to helix a3 of QOR. all three proteins, whereas the C-termini do not. A ‘‘he-lix breaking’’ residue is present at the kink (GlyLADH and GDH have taken different strategies to

avoid this collision. GDH does not have an equivalent 175:LADH, Pro 156:GDH, Gly 123:QOR, Pro 132:z-crystallin). There seems to be no preference for prolineto helix a3 (QOR), having instead a short, direct pep-

tide and a shorter helix a4 (Fig. 5). LADH has an equiv- or glycine in this position, suggesting that there isstructural conservation without sequence conserva-alent to helix a3 and a shortened helix a4. However,

the LADH a3 turns in the direction opposite to that of tion.QOR, resulting in a significant displacement of the he-lix relative to a3 in QOR. Further, the presence or Functional Considerations: Nucleotide Bindingabsence of helix a3 affects the position of the C-termi-

The bab nucleotide-binding regions for QOR andnal end of helix a2. Helix a2 is kinked in all threeLADH are depicted in Fig. 6. Structurally based studieson sequence patterns found in the fingerprint region ofthe nucleotide-binding domain of a number of NAD(P)-TABLE IIbinding proteins (29, 30) have identified different re-

Solvent-Accessible Surface Areas for the QOR, LADH, quirements for nucleotide specificity. NAD requires aand GDH Monomers GXGXXG sequence motif plus a negatively charged res-

idue at the end of strand bB, whereas specificity forTotal accessible Substrate-binding Area covered by

NADP is achieved by a GXGXXA sequence motif andsurface area site area Zn binding loopa positively charged residue near the end of strand bB.(A2) (A2) (A2)It appears that the nucleotide-binding motifs for QOR

LADH 14,300 119 1600 and z-crystallin are somewhat unusual since both en-GDH 15,300 139 920 zymes have single residue insertions in the region be-QOR 12,800 244 —

tween the first and the second conserved glycines. QOR


180 EDWARDS ET AL.

FIG. 6. The bab (bA, aB, bB) nucleotide-binding region showing the nucleotide-binding motif (labeled) and bound cofactor for (a) QORand (b) LADH. LADH has the classic GXGXXG nucleotide-binding motif, whereas QOR has a single residue insertion (Ala 149) to give anAXXGXXG motif. The different conformations for NADPH (QOR) and NADH (LADH) are clearly seen. The important residues, Val 172(QOR) and Asp 223 (LADH), at the end of strand bB, are also labeled.

has an AXXGXXG motif (Fig. 6a); z-crystallin has GXX- 2). This insertion maximizes contact between QOR andthe adenosine moiety of the cofactor. The insertion ofGXXG. Neither enzyme has the last alanine residue in

the motif predicted for NADPH binding, confirming the an extra residue (Ala 150) in the QOR sequence ap-pears to accommodate the adenine ribose phosphateproposal by Baker and coworkers (29) that this alanine

residue is not a prerequisite for NADPH binding. group by widening the cleft in this area. The widenedcleft in QOR leads to less direct contact between theVarious LADH/NAD complexes have been reported

(5). Table III lists the residues for QOR and LADH cofactor and QOR with a chain of water molecules beingused to mediate interactions between enzyme and co-which are involved in cofactor binding. Although struc-

turally equivalent residues are present, they are not factor.QOR has a small hydrophobic residue, Val 172, in-necessarily involved in cofactor binding. The structures

of QOR/NADPH and LADH/NADH show some notable stead of the larger negatively charged residue (Asp 223)required for NADH specificity in LADH (31). This mu-differences (Fig. 6). NADPH when bound to QOR is

bent and more compressed than NADH when bound tation is essential in providing sufficient space for theadditional adenine ribose phosphate group of theto LADH. The region of adenosine binding in QOR is

different from the corresponding region in LADH with NADPH cofactor. QOR has two positively charged resi-dues, Lys 177 and Arg 317, which surround the phos-the adenine ring being syn for QOR:NADPH and anti

for LADH:NADH (5). In QOR the loop between bE and phate group and stabilize the negative charge on thephosphate by electrostatic interactions. The interactionaF has moved closer to the cofactor to interact with the

adenine ring. QOR is also alone among these three with Lys 177 was easily predicted based on the require-ment for a positively charged residue at the end ofenzymes in possessing two serine residues (Ser 241 and

Ser 242) inserted into the sequence immediately after strand bB (30). The refined structure of QOR hasshown, however, that Arg 317 is also of importance inthe conserved region which includes strand bE (Fig.



TABLE III His 317, which may interact with the phosphate group.This residue represents an insertion between the QORComparison of Cofactor-Binding Residues

for LADH and QOR residues Glu 315 and Ser 316. These histidine residueswill be less flexible than the lysine and arginine QOR

QOR/NADPH LADH/NADH Conserved region? counterparts. The greater adaptability in this regionmay allow QOR to bind both NADPH and NADH while

Gly 173 Ile 224 N the relative inflexibility of z-crystallin limits it to(Val 217) Ile 269 Y

NADPH specificity.(Arg 219) Arg 271 YAla 148* Gly 199 YGly 151* Gly 210 Y Functional Consideration: The Substrate Binding SiteGly 152 Gly 202 Y(Val 172) Asp 223 Y Despite the high structural conservation in the re-Lys 177 Lys 228 N gion of the active site (helices a1 and a2 and strand b1),Phe 42 Arg 47 Y

there are major functional differences between theseGly 321* Arg 369 N(Ile 43) Ser 48 Y enzymes. The most significant is that QOR does notTyr 46 His 51 Y possess a metal equivalent to the catalytic zinc of(Thr 127) Thr 178 Y LADH and GDH and must therefore have a differentVal 153 Val 203 Y

mechanism for catalysis. LADH has a hydrophobic(Asn 240) Val 294 Ybinding pocket (7), whereas GDH possesses a chargedAsn 41* Cys 46 Y

(Leu 123) Cys 174 Y binding site (Rossjohn et al., unpublished results). TheLeu 266* Phe 319 N substrate-binding site for QOR [identified from thePhe 238 Val 292 Y crystal structure (9) and based on analogy to LADH]Pro 264 Ala 317 Y

has charged and neutral regions.Ser 216 (Val 268) YTable IV details the residues that line the substrate-Ser 241a — N

Tyr 130 (Gly 180) Y binding site in LADH and GDH and their equivalentTyr 192 (Pro 244) Y residues in QOR and z-crystallin. A comparison ofArg 317 (Gly 365) N

Note. Residues marked (*) do not directly interact with the cofactorbut make water-mediated contacts. Residues in parentheses do not TABLE IVinteract with the cofactor.

Residues Lining the Substrate-Binding Sitesa QOR:Ser 241 is in an insertion region and has no equivalent inLADH. for QORa z-Crystallin, LADH, and GDH

QOR z-Crystallin LADH GDH

providing electrostatic interactions to the phosphate — — Zn ZnAsn 41 Asn 48 Cys 46b Cys 40bgroup. Inspection of the structure strongly suggestsThr 63 Thr 71 His 67b His 67bthat QOR should show NADPH specificity, but it ap-Leu 123 Ile 131 Cys 174b Glu 155b

pears to be active with both NADPH and NADH (Lilley— — Leu 116c —

et al., unpublished results). — — Phe 93 Val 92The above observations may provide an explanation — — Leu 141c —

— — Phe 140c —for the NADPH specificity shown by z-crystallin. z-Ile 43 Val 50 Ser 48d Thr 42d

Crystallin has a GXXGXXG binding motif. The firstAsn 240 Cys 248 Val 294 Thr 276glycine (Gly 156) in the motif may be less versatile Tyr 52 — Leu 57 —

than the alanine of QOR. Inspection of the sequence Ser 265 Ser 270 Ile 318 Ser 303alignment suggests that Lys 187 of z-crystallin may Leu 266 Leu 271 Phe 319e Val 304e

Arg 263 Gly 268 Gly 316e Ala 301ebe equivalent to Lys 177 of QOR. Examination of thesequence alignment with concomitant visualization of

Note. A dash denotes no equivalent residue.the structure of QOR suggests that this residue is not a The potential substrate-binding site for QOR has been identifiedin a suitable position for interaction with the phosphate based on the X-ray structure (9), analogy to LADH, and biochemicalgroup. z-Crystallin:His 200, equivalent to QOR:Tyr considerations.

b Residues which bind the catalytic zinc in LADH and GDH.192, may provide electrostatic interactions with thec Residues which are part of the structural zinc-binding loop inphosphate group. Unlike QOR, which has a flexible

LADH.arginine (Arg 317) in the tight turn between b12 and d Catalytic residues in LADH and GDH.a5, z-crystallin has a serine residue at this position. z- e Equivalent residue in LADH and GDH but not part of substrate-

binding site.Crystallin does, however, have a possible alternative,


182 EDWARDS ET AL.

structurally equivalent residues to QOR shows marked LADH and we anticipate that such a framework maybe used to accurately model the structures of otherdifferences in the nature of the residues surrounding

the pocket. Residues Cys 46, His 67, and Cys 174 which MDR enzymes. Predictions based solely on multiplesequence alignments may be misleading. Comparisonsbind the catalytic zinc in LADH have been replaced by

Asn 41, Thr 63, and Leu 123 in QOR. A similar set of of the crystal structures of several members of an en-zyme family may therefore provide greater insight andresidues is found for z-crystallin. Further, the catalytic

residue which binds the hydroxyl group of the alcohol understanding of the structure–function relationshipswithin an enzyme family and result in improved multi-or glucose substrate, Ser 48:LADH and Thr 42:GDH,

has been replaced by a hydrophobic residue in both ple sequence alignments.QOR and z-crystallin (Ile 43 and Val 150, respectively).There is only one other member of the MDR superfam- ACKNOWLEDGMENTSily [the predicted protein product of the Trichoderma

J.R. is supported by a Royal Society Fellowship (1995) and thanksharzianu indc11 gene (4)] which shares this replace- M. J. Danson and D. W. Hough for the GDH project. We also thankment. In LADH the basic residue, His 51, is involved N. E. Dixon for his contribution to the QOR project.in a proton shuttle with Ser 48 and O2*A of the boundNADP. The equivalent residue in QOR, Tyr 46, is also

REFERENCESbound to the nicotinamide ribose sugar, but there is1. Persson, B., Krook, M., and Jornvall, H. (1991) Eur. J. Biochem.no possibility of a similar proton shuttle mechanism

200, 537–543.operating in QOR, as Ile 43:QOR is unable to make2. Scopes, R. K. (1983) FEBS Lett. 156, 303–306.hydrogen bond contact with the nicotinamide ribose.3. Jornvall, H., Persson, B., and Jeffrey, J. (1987) Eur. J. Biochem.In GDH, the equivalent residue to His 51:LADH is Gly

167, 195–201.45:GDH. The glycine at this position allows the oxygen4. Persson, B., Zigler, J. S., and Jornvall, H. (1994) Eur. J. Biochem.atom of Thr 42:GDH to transfer a proton directly to 226, 15–22.

bulk solvent. The substrate-binding site of z-crystallin5. Eklund, H., Samama, J-P., and Jones, T. A. (1984) Biochemistry

is similar to that of QOR, having four strictly conserved 23, 5982–5996.residues and two conservative changes. The most nota- 6. Ramaswamy, S., Eklund, H., and Plapp, B. V. (1994) Biochemis-ble differences between QOR and z-crystallin are found try 33, 5230–5237.at Arg 263, where the equivalent residue in z-crystallin 7. Eklund, H., Nordstrom, B., Zeppezauer, E., Soderlund, G., Ohls-

son, I., Boiwe, T., Soderbury, B-O., Tapla, O., Branden, C-I., andis a glycine (Gly 268), and at Tyr 52 where there isAkeson, A. (1976) J. Mol. Biol. 102, 27–59.no equivalent z-crystallin residue. No residues in the

8. Edwards, K. J., Thorn, J. M., Daniher, J. A., Dixon, N. E., andsubstrate binding site are conserved between QOR andOllis, D. L. (1994) J. Mol. Biol. 240, 501–503.LADH, and only one residue, Ser 265:QOR, is con-

9. Thorn, J. M., Barton, J. D., Daniher, J. A., Dixon, N. E., Ollis,served between QOR and GDH. This gives the strong-D. L., and Edwards, K. J. (1995) J. Mol. Biol. 249, 785–799.est indication of the divergent nature of these enzymes.

10. John, J., Crennel, S. J., Hough, D. W., Danson, M. J., and Taylor,G. L. (1994) Structure 2, 385–393.

11. Rao, P. V., Krishna, C. M., and Zigler, J. S. (1992) J. Biol. Chem.Concluding Remarks267, 96–102.

These structurally based comparisons of three MDR 12. Rao, P. V., and Zigler, J. S. (1992) Biochim. Biophys. Acta 1117,enzymes, QOR, GDH, and LADH, confirm that despite 315–320.low sequence identity they possess a high degree of 13. Sun, H-W., and Plapp, B. V. (1992) J. Mol. Evol. 34, 522–535.homology in their overall fold. This is true not only for 14. Jornvall, H., Persson, B., Du Bois, G. C., Lavers, G. C., Chen,

J. H., Gonzalez, P., Rao, P. V., and Zigler, J. S., Jr. (1993) FEBSthe nucleotide-binding domain but also for the catalyticLett. 322, 240–244.domain. This study presents a classic example of diver-

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16. Bernstein, C. F., Koetzle, T. F., Williams, G. J. B., Meyer, E. F.,within the amino acid sequences a stable enzyme foldJr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T.,has been retained. The catalytic zinc ion has been lost and Tasumi, M. (1977) J. Mol. Biol. 112, 535–542.

in QOR and z-crystallin, and those residues important 17. Al-Karadaghi, S., Cedergren-Zepperzauer, E. S., and Hovmoller,for catalysis have been significantly altered to accom- S. (1994) Acta Crystallogr. D50, 793–807.modate the new activity of the enzyme. 18. Brunger, A. T. (1992) X-PLOR Version 3.1 Manual: A System

We propose that the structure of z-crystallin is com- for X-Ray Crystallography and NMR, Yale Univ. Press, NewHaven, CT.parable to that of QOR and that z-crystallin has a simi-

19. Lee, B., and Richards, F. M. (1972) J. Mol. Biol. 55, 379–400.lar active site. A structural framework for the MDR20. Jones, T. A. (1985) Methods Enzymol. 115, 157–171.superfamily has been proposed based on the structur-

ally conserved regions common to QOR, GDH, and 21. Connolly, M. L. (1983) Science 221, 709–713.



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Documents

Structural and Sequence Comparisons of Quinone Oxidoreductase, ζ-Crystallin, and Glucose and Alcohol Dehydrogenases