The Reaction Mechanism of Phospholipase D from Streptomyces sp. Strain PMF. Snapshots along the Reaction Pathway Reveal a Pentacoordinate Reaction Intermediate and an Unexpected Final

The Reaction Mechanism of Phospholipase D fromStreptomyces sp. Strain PMF. Snapshots along theReaction Pathway Reveal a Pentacoordinate ReactionIntermediate and an Unexpected Final Product

Ingar Leiros1*, Sean McSweeney2 and Edward Hough1*

1Department of ChemistryFaculty of Science, Universityof Tromsø, Tromsø, Norway

2EMBL c/o ILL, F-38043Grenoble Cedex, France

Almost all enzyme-catalysed phosphohydrolytic or phosphoryl transferreactions proceed through a five-coordinated phosphorus transition state.This is also true for the phospholipase D superfamily of enzymes, wherethe active site usually is made up of two identical sequence repeats of anHKD motif, positioned around an approximate 2-fold axis, where the his-tidine and lysine residues are essential for catalysis. An almost completereaction pathway has been elucidated by a series of experiments wherecrystals of phospholipase D from Streptomyces sp. strain PMF (PLDPMF)were soaked for different times with (i) a soluble poor, short-chainedphospholipid substrate and (ii) with a product. The various crystal struc-tures were determined to a resolution of 1.35–1.75 A for the differenttime-steps. Both substrate and product-structures were determined inorder to identify the different reaction states and to examine if the reactionactually terminated on formation of phosphatidic acid (the true product ofphospholipase D action) or could proceed even further. The results pre-sented support the theory that the phospholipase D superfamily shares acommon reaction mechanism, although different family members havevery different substrate preferences and perform different catalytic reac-tions. Results also show that the reaction proceeds via a phosphohistidineintermediate and provide unambiguous identification of a catalytic watermolecule, ideally positioned for apical attack on the phosphorus and con-sistent with an associative in-line phosphoryl transfer reaction. In one ofthe experiments an apparent five-coordinate phosphorus transition stateis observed.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: pentacovalent phosphorus; transition state; reactionmechanism; X-ray crystallography; phosphohistidine*Corresponding authors

Introduction

Phospholipase D (PLD; EC 3.1.4.4) catalyses thehydrolysis of phospholipids (normally phospha-tidylcholine) into phosphatidic acid (PA) and apolar head group (normally choline). In additionto this hydrolytic activity, PLD also catalyzes atransphosphatidylation reaction in vitro to formnew phospholipids. Even at very low levels (1%)of ethanol, transphosphatidylation can bepreferred, resulting in the almost exclusiveproduction of phosphatidylethanol rather than PAand choline.1 This reaction is unique for PLDenzymes and thus provides an unambiguousindication for the presence of PLD activity. In

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

Present address: I. Leiros and S. McSweeney,Molecular Crystallography Group, ESRF, BP220, F-38043,Grenoble Cedex, France.

E-mail addresses of the corresponding authors:[email protected]; [email protected]

Abbreviations used: PLD, phospholipase D; PLDPMF,phospholipase D from Streptomyces sp. strain PMF; PLC,phospholipase C; Tdp1, tyrosyl-DNAphosphodiesterase; PSS, phosphatidylserine synthase;Nuc, endonuclease from Salmonella typhimurium; DAG,diacylglycerol; PA, phosphatidic acid; MAD,multiwavelength anomalous dispersion.

doi:10.1016/j.jmb.2004.04.003 J. Mol. Biol. (2004) 339, 805–820

addition to short-chained alcohols, lipids can beused as acyl acceptors in the transphosphatidyla-tion reaction resulting in even more complex lipidproducts. As an example, PLD from cabbage cansynthesize cardiolipin (diphosphatidylglycerol)from two phosphatidylglycerol molecules in atwo-step process starting with PLD hydrolysis ofone phosphatidylglycerol molecule into PA andglycerol, followed by PLD transphosphatidylationon the second molecule of phosphatidylglycerol,with PA as the acyl acceptor.2 It has also beenshown that the uncommon and interesting bis-phosphatidic acid can be formed by the combinedactions of phospholipase C (PLC) and PLD on twomolecules of phosphatidylcholine. The reaction isinitiated by PLC hydrolysis of one of the phospha-tidylcholine molecules into diacylglycerol (DAG)and phosphorylcholine, followed by a trans-phosphatidylation reaction catalyzed by PLD,where DAG acts as an acyl acceptor to formbisphosphatidic acid and choline.3

Catalytic reaction and substrate specificity

More than 30 years ago, Stanacev & Stuhne-Sekalec,2 proposed that the catalytic reactionfor PLD enzymes proceeds via a covalentphosphatidyl-enzyme intermediate. A study ofincorporation of H2

18O into distearoyl-phospha-tidylcholine during PLD hydrolysis,4 indicatedthat the catalytic reaction of PLD from Streptomyceschromofuscus proceeds through P–O rather thanC–O bond cleavage. Other studies indicated thatPLD from cabbage retained the configuration ofthe phosphorus atom in both the hydrolysis andin the transphosphatidylation reaction withdifferent radioactively labelled substrates, allhaving a chiral phosphorus atom.5,6,7 In a recentpaper, a comparison between PLD activity fromtwo Streptomyces species is presented.8 Interest-ingly, one of these PLD enzymes possesses neitherof the above-mentioned HxK(x)4D (HKD) sequencerepeats, resulting in an apparently altered reactionmechanism, which does not proceed through acovalent enzyme–substrate intermediate.8

This HKD sequence motif has been found in avariety of enzymes having a very widespreadrange of activity, including phospholipase D, endo-nucleases, phosphatidylserine synthases (PSS),cardiolipin synthases, as well as proteins having aso far unknown in vivo function.9,10,11 The commondenominator of proteins belonging to this PLDsuperfamily of enzymes is that they all performcatalysis involving a phosphorus atom.

Phosphatidylserine synthase (PSS) has, asmentioned, been classified as a PLD superfamilymember. When radioactive labelling experimentswere performed on PSS from two different species,opposing results were obtained. PSS fromEscherichia coli acted with net retention of configur-ation of the phosphorus atom, while PSS fromyeast acted through a different reaction mechan-ism, which was claimed to result in an inversion

of configuration.12 However, as already mentionedfor PLD, only the PSS from E. coli possesses theconserved HKD sequence motif, again suggestingan explanation for the observed differing reactionmechanisms. The conclusion therefore is that mostPLD-, as well as E. coli PSS-catalyzed reactionsproceed through a two-step ping-pong mechanism,involving the formation of a phosphatidyl-enzymeintermediate.

The presumed covalent phosphatidyl-enzymeintermediate was thought to be formed betweenPA and a reactive amino acid residue in theenzyme. Mutational studies on human PLD1(hPLD1) indicated a catalytically critical serineresidue (Ser911), which has also been suggested toform a covalent phosphatidylserine intermediate.13

Other studies aiming to identify residues involvedin the catalytic reaction have been performed,11,13,14

and in bacterial PLDs, the main catalytic residuesare thought to be two histidine residues (His170and His448 in PLD from Streptomyces sp. strainPMF (PLDPMF)).15 The crystal structure ofrecombinant Nuc, an endonuclease from Salmonellatyphimurium and the smallest known member ofthe PLD superfamily also indicated that thecatalysis involves two histidine residues.16 Nuc iscrystallized as a homodimer, and it is most likelyalso active in solution as a multimeric protein.17

The active site in the crystal structure of Nuc islocated on the interface between two monomers,with identical residues from two crystallographi-cally related molecules forming one active site.16

The reported crystal structure of a third memberof the PLD superfamily, human tyrosyl-DNAphosphodiesterase, Tdp118 shows this phospho-hydrolase to have a similar fold to PLDPMF andNuc. In contrast to Nuc but similar to PLDPMF,Tdp1 is a monomer with an approximate 2-foldsymmetry axis through the active site. The activesites of all three enzymes are similar, includingstructural conservation of the important residues(numbering according to the crystal structure ofPLDPMF) His170 and His448, Lys172 and Lys450,and Asn187 and Asn465, as well as conservativemutations of the histidine-coordinating residuesAsp202 and Asp473 to Gln and Glu, respectively,in Tdp1 and Glu in Nuc. Another member of thePLD superfamily, the 586 amino acid residueYersinia pestis murine toxin (Ymt),19 is claimed tobe active as a dimer20 although the monomer itselfcontains two of the HKD sequence motifs. Theenzymatic reaction catalyzed by Ymt has alsobeen suggested to proceed via the formation of acovalent enzyme–substrate–intermediate.14,20

Although both Nuc, Tdp1 and Ymt are capable ofbinding and hydrolyzing a phosphodiester moiety,they all have a biological function very unlikePLD. Nuc is an endonuclease, which cleaves bothsingle- and double-stranded DNA.21 The PLDsuperfamily member Tdp1 catalyzes the hydrolysisof a phosphodiester bond between a tyrosineresidue and a DNA 30 phosphate. The in vivofunction of Tdp1 has been proposed to be the

806 The Reaction Mechanism of Phospholipase D

hydrolysis of the protein–DNA linkage caused bystalled topoisomerase I on DNA.18 Recently, anapparent substrate intermediate mimic of amixture of DNA, peptide and vanadate hasbeen trapped covalently linked to an active sitehistidine (His263) from the N-terminal domain ofTdp1.22

Ymt, on the other hand, is a PLD superfamilymember with an undefined function, except that itis said to be important in order for Y. pestis tosurvive and spread. There are no known nativesubstrates for Ymt, except that a study hasreported Ymt to hydrolyze the terminal phos-phodiester bond in several phospholipids,with preference for phosphatidylethanolamineover phosphatidylcholine and phosphatidyl-serine.20

Although crystal structures have been deter-mined and reaction mechanisms have been studiedfor several PLD superfamily members, there is stilllittle structural knowledge about the nature of(phospholipid) substrate binding in the active siteof the PLD enzymes themselves. Amino acidresidues important for catalysis have been identi-

fied, but so far only the nature of the enzyme-inter-actions with the phosphorus moiety of thesubstrate has been identified. PLDPMF has beenfound to accommodate a wide range of phospho-lipids as substrates, with broad head groupspecificity.23 Information about the binding andorientation of the substrate phospholipid in theactive site would potentially explain the specificityof PLD enzymes between the different substrates.

Results

Presented structures

Structure 1: unliganded PLDPMF

The active site and its environment are similar tothat presented for the endonuclease, Nuc, in itstungstate-free form,16 and to the crystal structureof unliganded Tdp1.18 The main difference is thatthe two aspartate residues (Asp202 and Asp473),which form strong hydrogen bonds to the twohistidine residues in the active site of PLDPMF aresubstituted by glutamate residues in Nuc and a

Figure 1. The active site of PLDPMF unliganded or phosphate-inhibited. (a) The native state (structure 1). Two watermolecules (OW1 and OW2) occupy the binding site for the phosphate moiety. (b) Phosphate-inhibited PLDPMF

(structure 2). Both electron density maps are sA-weighted 2mFo 2 DFc maps contoured at 1.5s.

The Reaction Mechanism of Phospholipase D 807

glutamine and a glutamate in Tdp1. Two active sitewater molecules are observed in a bridged positionbetween the mentioned His170 and His448 (Figure1(a)).

Structure 2: PLDPMF–PO4

This structure has been presented.15 The findingsare briefly summarized in the following. Crystalsof PLDPMF are normally grown in a buffer contain-ing 0.1 M citrate–phosphate and the identificationof a tetrahedrally coordinated phosphate-ion(probably HPO4

22 at pH 5.4) in the active site ofthe protein was not unexpected. Both phosphateand tungstate have been shown to be competitiveinhibitors for PLDPMF at low concentrations(S. Servi, personal communication). With oneexception, the active site environment in the phos-phate-inhibited structure of PLDPMF, is differentfrom all the other crystal structures in the presentstudy, in that one of the active site residues,His170 is rotated towards the entrance of the activesite, leaving the side-chain oxygen atoms of thenegatively charged Asp473 in an unbound state.This position for His170 is referred to as the“external“ and very likely inactive conformation,whereas the position where His170 is forming anion-pair interaction to Asp473 is generally referredto as the “internal” conformation (Figure 1(b)).

Structure 3: PLDPMF–WO4

This structure arises from the remotewavelength (0.89 A) of a multiwavelengthanomalous dispersion (MAD) data set processedto 1.7 A resolution. During the initial refinementwith both the active site His170 and the tungstenatom omitted, it became evident that the positionand coordination of the tungsten atom in the activesite was somewhat unusual. An octahedraltungsten moiety was built into electron densitywhere one of the apical ligands to the tungstenatom is the N12 side-chain atom of His170. In thisstructure, the side-chain of His170 is located in theabove-mentioned “internal” conformation, unlikethe phosphate-inhibited structure, with a refinedW–N distance of 2.19 A. The active site region ofthis structure is comparable to the reported crystalstructure of tungstate-inhibited Tdp1 (pdb1mu7)where the active-site tungsten atom is also foundin an octahedral conformation with a W–Ndistance of 2.33 A. The similar crystal structure ofthe tungstate-inhibited Nuc (pdb1bys) shows thetwo symmetrical W–N distances in the active siteof Nuc to be considerably longer (2.9 A) than forboth Tdp1 and PLDPMF. However, the distancebetween the other histidine in the active site,His448 and the tungsten atom in PLDPMF is 3.46 A(the similar distance is 3.61 A in tungstate-inhibited Tdp1), i.e. considerably longer than inNuc. As the active site of Nuc is made up of twocrystallographically related monomers, with acrystallographic 2-fold axis running through it, the

two histidine residues in the active site of Nuc areindistinguishable and it is therefore not possible toassign individual functions for the two residues.Observing the octahedral state of the tungstenatom in PLDPMF was unexpected, as was the find-ing that two of the equatorial ligands haveadditional electron density attached to them,causing a compression of one of the equatorialO–W–O angles from the expected 908 down to69.48, which is somewhat similar to what has beenobserved (828) for tungstate-inhibited Tdp1.24 It isnot possible to assign the chemical nature of thisequatorial ligand in PLDPMF, although it appearsto be similar to an ethylene glycol moiety (Figure 2).

Structures 4, 5 and 6: substrate (diC4PC) soaks

The datasets representing structures 4, 5 and 6were collected at different time steps along thereaction pathway of PLDPMF using 2–3 mMdibutyrylphosphatidylcholine (diC4PC) as sub-strate. The data sets were collected from differentcryo-cooled crystals after 30 minutes, eight hours,and eight days of soak. The soaking time wasbasically determined by the availability of synchro-tron beamtime, as well as the fact that a soakingtime shorter than 30 minutes resulted in poordiffraction. The latter was assumed either to be aconsequence of substrate diffusion into thecrystals, or due to an ongoing reaction. At the con-ditions used, PLDPMF should be fully active, andhas been shown to have a broad pH optimumranging from 4.0 to 6.0.25 (Figures 3, 4 and 5(a)).

Structure 4: 30 minute soak. Structure 4 representsthe data collected after the shortest possible soak-ing time giving crystals suitable for data collectionpurposes. Electron density maps after an initialrefinement with His170 and all water molecules inor near the active site omitted from the refinement,showed the presence of the covalently attachedcatalytic product in the active site. After the firstmanual building round, His170 and a single phos-phorus atom was built into electron density andincluded in refinement. Figure 3(b) shows theresulting electron density around His170 with den-sity from neighbouring protein residues omittedfor clarity. Both the sA-weighted 2mFo 2 DFc andmFo 2 DFc maps indicated the presence of anapparently pentacoordinate phosphate withfurther electron density associated with one of theequatorial oxygen atoms pointing towards theentrance of the active site. The latter electron den-sity is due to the DAG moiety of the product PAand although flexible, this mainly hydrophobicpart of the product could be fitted into electrondensity at a low map contour level (0.7s in the2mFo 2 DFc map) and thereafter included in refine-ment. This structure represents the intermediatestate where the product (PA) is still attached to theenzyme through a covalent bond and an activatedwater molecule has approached the phosphorusand is sufficiently close to this phosphorus to bein a pentacoordinated state where both apical


ligands have considerable bond character (i.e. thatthe P–O distances are much shorter than the vander Waals distance) and the other ligands areclearly equatorial to the phosphorus. The firstproduct, choline, which is hydrolyzed subsequentto the nucleophilic attack by His170 has diffusedout of the active site. Based on this intermediatestructure, the choline moiety was modelled intothe active site of PLDPMF (not shown), in order toindicate the positioning of the head group andpossibly illuminate why the bacterial PLDenzymes have a particularly broad specificitytowards the chemical character of the head-groupof the substrate phospholipid.

Structure 5: eight hour soak. Structure 5 is theresult of an eight hour soak with diC4PC, showingthe side-chain of His170 in both the “internal” and“external” conformations (Figure 4). In the internalconformation, where His170 Nd1 forms an ion-pairinteraction to the side-chain of Asp473, a residualelectron density is assigned as phosphohistidine,coinciding with what is observed for structure 6,i.e. the eight day substrate soak presented below.A water molecule is observed in an apical position

to the phosphorus atom at a P–O distance of4.02 A. As already mentioned, this water moleculewould be ideally positioned to act as the secondnucleophile in the hydrolytic reaction releasingthe product PA. In the external conformation,additional difference density can be seen whichcould potentially be the product PA re-enteringthe active site, which will be discussed in moredetail later.

Structure 6: eight day soak. The first data setcollected in this series was actually set 6, i.e. afteran eight-day soak. sA-Weighted26 mFo 2 DFc and2mFo 2 DFc electron density maps revealed a tet-rahedral phosphorus atom covalently attached tothe N12 side-chain atom of His170, forming aphosphorylated histidine, with a refined P–Ndistance of 1.67 A and P–O distances of 1.50–1.56 A. In addition to this, a water molecule wasobserved in an apical position to the phosphorus,at a P–O distance of 3.79 A (Figure 5(a)), ideallypositioned for an in-line attack of the phos-phorus. As a consequence of these observations,a more thorough search for intermediate stepson the reaction pathway was initiated, resulting

Figure 2. Tungstate-inhibited PLDPMF (structure 3). (a) Only the tungsten atom and the side-chain of His170are included in the refinement. The octahedral state of the tungsten ligand is clearly visible in the differencedensity maps. (b) As (a), but all the five oxygen ligands to the tungsten atom are included in the refinement as well.The 2mFo 2 DFc and mFo 2 DFc maps are contoured at 1.5s and 4s, respectively.


in the above-mentioned data sets collected at soaktimes of 30 minutes and at eight hours.

Structures 7 and 8: product (glycerophosphate)soaks

The observations from the substrate-soakingexperiments indicate that, at least in the crystals,

covalently attached phosphate is the stable end-step of the reaction. This could be explained eitherby re-entry of the product into the active site withsubsequent cleavage of the DAG backbone andformation of a phosphohistidine, or by the loss ofthe DAG moiety as a consequence of “ageing” asthe crystals were soaked for a relatively long time.The datasets 7 and 8, obtained after soaking with theproduct-like compound, glycerophosphate (the

Figure 3. Substrate soaking experiments using diC4PC soaked for 30 minutes (structure 4). (a) An illustration of thechemical structure of diC4PC. (b) The active site region using only the phosphorus atom in the refinement. Differencedensity describing the pentacovalent phosphorus atom can be seen. Maps are contoured at 1.0s and 3.0s for the2mFo 2 DFc and mFo 2 DFc maps, respectively. (c) Same as (b), but refined with a five-coordinated phosphohistidinewith the complete lipid backbone included in refinement. Maps are contoured at 1.0s and 4.0s for the 2mFo 2 DFc

and mFo 2 DFc maps, respectively. For clarity, neighbouring residues have been omitted in this view; these are,however, oriented similarly to all the other structures.


minimal “phospholipid” product, having only theglycerol framework), were collected after 30 min-utes and 90 minutes of soaking time, respectively.Both structures clearly indicate the presence ofphosphate covalently attached to His170 in theactive site. This confirms the assumption that thePA product of the primary reaction indeed can re-enter the active site to be processed into a DAGmoiety (in this case glycerol) and phosphorylatedenzyme. In addition, the catalytically crucial watermolecule (OW in all Figures) refines to a positionapical to the P–N bond in the phosphohistidine.Electron density associated with His170 and thiswater molecule for the structures 7 and 8 is shownin Figure 5.

Discussion

Enzyme-catalyzed phosphoryl transfer reactionscan proceed in several ways,27 but in general thechoice is between an SN1-type dissociative path-

way where the leaving group exits before thenucleophile attacks the phosphorus to form ametaphosphate (tricoordinate) reaction inter-mediate, uncharged in the case of phosphodiestersubstrates; or an SN2-type associative pathway,where a nucleophile attacks the substrate beforethe leaving group departs to form a pentacoordi-nated negatively charged reaction intermediate. Inthe associative pathway the reaction intermediateis approximately planar trigonal bipyramidal withtwo apical ligands, while in the dissociative path-way, the trigonal planar structure is maintainedwithout coordination to apical ligands. Thedifference in charge for the two intermediates, thepresence of divalent metal ions, positively chargedresidues and/or hydrogen bond donors distri-buted around the equatorial oxygen atoms of thereaction intermediate should generally favor thepentacoordinate intermediate. The enzymaticmechanism can also be deduced from the state ofthe transition state and from the stereochemistryof the reaction products.

Figure 4. Substrate soaking experiments using diC4PC soaked for eight hours (structure 5). (a) Refined without aphosphate moiety or the second conformation of His170 included. (b) Including the phosphohistidine and second con-formation of His170 with an occupancy of 0.4. In both presentations, the water molecule critical for catalysis (OW)refined to the same position. Maps are contoured at 1.0s (2mFo 2 DFc in blue) and 4.0s (mFo 2 DFc in red).


Five-coordinate phosphate

In the present study, the 30 minutes substratesoak (structure 4) reveals the nucleophilic attackby a water molecule to release the product

diC4PA. There is a clear presence of a fifth ligandin an apical position on the phosphorus atom inboth mFo 2 DFc and 2mFo 2 DFc electron densitymaps (Figure 3). The refined bond distances are1.83 A and 1.96 A for the P–N (leaving group)

Figure 5. Illustration of the soaking experiments where the stable “end-point” of the reaction is reached. (a) Substrate(diC4PC) soaked for eight days (structure 6). (b) Product (glycerophosphate) soaked for 30 minutes (structure 7).(c) Same as (b) but soaked for 90 minutes (structure 8). All maps are sA-weighted 2mFo 2 DFc maps contoured at1.5s. The distance shown is the distance between the phosphorus and the catalytic water molecule in the apicalposition to the phosphorus.


and P–O (nucleophile) interactions, respectively,although the mFo 2 DFc difference map when onlythe phosphorus atom is included in refinement(Figure 3(b)) indicates the latter distance to besomewhat longer, i.e. 2.1–2.15 A. The refinementtarget value for the P–N bond was 1.85 A and theestimated standard deviation weighting wasrelaxed from the default of 0.02 A to 0.1 A. For theP–O bond, the target value was 1.95 A with aweight of 0.1 A. The refined P–N bond length isrelatively close to what is observed for the P–Nbonds in 1,3-diphosphorylimidazole (average of1.78 A),28 while the apical P–O distance, althoughlonger than the normal covalent single bond length(1.55–1.6 A), is much shorter than the van derWaals distance (3.4 A). This illustrates the associat-ive mechanism in action, indicating a substantialbond character between the phosphoryl groupand the donor and acceptor groups at the sametime. Whether the structure actually shows thetransition state is perhaps a question of definition,but a closely related situation has recently beenreported for an a-thrombin–phosphonatecomplex.29 In that case the enzyme was soaked forseven days at 4 8C before data collection and theauthors argue that the pentacoordinate state hasbeen stabilized by interactions in the active sitecombined with the fact that the ligand is a slowsubstrate. A longer soak (12 weeks) eventually ledto loss of two phenyl-ester groups and the for-mation of a phosphoryl analogue of the tetrahedraltransition state after attack by the catalytic Ser195(in a-thrombin). Hajdu and co-workers have pub-lished a very interesting study regarding the reac-tion rates in crystals compared to solution.30 Theyshow that different steps along the reaction path-way can be trapped in crystals even with data col-lected at room temperature and using substratesoaking periods in the scale of hours. It is alsoemphasized that in enzymatic reactions, depend-ing on the rate constants, there is a possibility of abuild-up of an intermediate if its formation is fasterthan its breakdown into product.30 In the case ofPLDPMF, a recent study has shown that the actualreaction rate for the turnover of the substratediC4PC into diC4PA in solution is in the order ofminutes, i.e. a few conversions per hour.8 Thesame authors also show that the specific activityof PLDPMF is heavily dependent on the substrateconcentration, and also that the enzyme appearsto exhibit moderate interfacial activation, i.e. it ismore active when embedded onto a membranethan in solution.8 All these observations contributeto explaining why a transition state intermediatemay be observed after a substantial soaking time.In the crystals, there appears to be a build-up ofthe reaction intermediate where the covalent inter-mediate between His170 and the reaction productdiC4PA is trapped in the active site of the enzyme.This would imply that the nucleophilic attack onthe phosphorus atom by the second nucleophile, awater molecule activated by His448 (illustrated inFigure 6, although this is for the phosphohistidine

end-step), is the rate-limiting step in the catalyticreaction (in the crystals). Related to this is the factthat in all crystal structures of PLDPMF, two surfaceloops (residues 126–129 and 382–389) aredisordered. Both of these are located on the rim ofthe active site cavity, and the latter in particular ishighly interesting, since its sequence pattern (382-RGAVGSGG-389) is highly conserved amongother bacterial PLD enzymes with HKD sequencemotifs. With the reported moderate interfacialactivation of PLDPMF,

8 there is a clear possibilitythat these loops are critical in embedding theenzyme onto a membrane to ease the diffusion ofsubstrate in and product out of the active site.

The initial nucleophile

Earlier studies have stated that the formation ofa covalently linked phosphohistidine moiety is themost probable reaction intermediate for enzymesin the PLD superfamily.14,20,30 It has also beenshown that mutations of either His or Lys residuesin the active site of Nuc,20 Ymt,14 or Tdp131 resultin an enzyme without the ability to hydrolyze aphosphodiester substrate. Mutational studies onhuman PLD1 (hPLD1)13 have shown the catalyticimportance of corresponding residues in thatenzyme, although a serine was suggested to formthe covalent phosphatidyl-intermediate. ForPLDPMF, chemical modifications have suggestedthat Lys and not His is essential for activity.32

From the crystal structure of PLDPMF, it is apparentthat, as indeed suggested as a possibility by theauthors, modification of the Lys residues couldwell protect the active site His residues againstchemical modification. The results of the presentstudy indeed confirm the catalytic importance ofthe two histidine residues in the active site.Furthermore, the results unambiguously identifywhich of these two active site histidine residues(His170 and not His448) in PLDPMF actually is thefirst nucleophile in the catalytic reaction. Iwasakiand co-workers33 proposed that a histidine residuein the C-terminal domain of PLD from Streptomycesantibioticus (His442 in that enzyme) was the residueinvolved in the formation of a covalent enzyme–substrate intermediate in the catalytic reaction.

In structure 5 (the eight hour substrate soak;Figure 4), the side-chain of His170 is present astwo rotamers, one of which is similar to that inphosphate-inhibited PLDPMF (structure 2), with theside-chain rotated away from the active site intothe external position and, as a consequence, notable to form the stabilizing hydrogen bond toAsp473. Since the external orientation points inthe direction where substrate enters and productexits the active site, this movement could beimportant in order to (i) guide the substrate intothe active site before catalysis takes place and/or(ii) guide the product out of the active site aftercatalysis has occurred. From Figure 4, it can beseen that there is residual electron density both inthe 2mFo 2 DFc and mFo 2 DFc electron density


maps when the side-chain of His170 is oriented inthis external conformation. On building His170into electron density in the external conformation,it becomes apparent that, in addition to a x1rotation, the side-chain is also flipped 1808 via ax2 rotation on moving from the internal to theexternal conformation. His170 Nd1 then forms ahydrogen bond to the main-chain oxygen inresidue Trp168 with a distance of 3.24 A. Theobservation that His170 has residual electron den-sity attached to it in the external conformationcould indicate that it is able to act as a nucleophilein this conformation, or that this residue is rotatedinto this conformation when the product is leaving.Both alternatives are possible, but mutationalstudies on hPLD1 have shown that upon mutationof Ser911 in that enzyme into Ala, activity wascompletely lost.13 The corresponding residue inPLDPMF is Ser463 (see Figure 6), where its Og atomforms a hydrogen bond to Asp473 Od1, in turnhydrogen binding to His170 Nd1 (when His170 isin the internal conformation). From loss of activityin hPLD1 when Ser is mutated into Ala, it seemslikely that His170 must be in the internal confor-mation in order for catalysis to occur.

In all crystal structures presented here except forthe phosphate-inhibited, His170 is oriented tomake a stabilizing ion-pair interaction to Asp473(His170 Nd1–Asp473 Od1 distance of about 2.5 A).In the published structure of the recombinant

endonuclease, Nuc,16 the orientation of the singlehistidine residue (His94) in the active site of Nucis such that a similar interaction is obtained. It isimportant to note that Nuc crystallizes as a dimerwith a crystallographic 2-fold axis running throughthe active site. This fixes the active site environ-ment, i.e. the two HKD motifs in the active site ofNuc are structurally identical. It is therefore notpossible to use the information obtained from thestructure of Nuc to assign potentially differentroles to the two histidine residues in the active siteof PLDPMF, for other PLD enzymes, or indeed forthe other enzymes in the PLD superfamily whichcontain two HKD motifs and are active as bi-lobalmonomers.

The crystal structure of human tyrosyl-DNAphosphodiesterase (Tdp1), a PLD superfamilymember in which the Asp in the HKD motifs isreplaced by Leu,18 shows this phosphohydrolaseto have a similar fold to PLD and Nuc. However,in contrast to Nuc but similar to PLDPMF, Tdp1 is amonomer with an approximate 2-fold symmetryaxis through the active site. The crystal structuresof vanadate- and tungstate-inhibited Tdp1,24 aswell as a crystal structure of an apparent transitionstate mimic with vanadate replacing phosphate inthe substrate analogue,22 have firmly establishedthat His263 (the structural analogue to His170 inPLDPMF) in the N-terminal domain of Tdp1 isresponsible for the first nucleophilic attack. This

Figure 6. A view of the active site region for one of the product soaks (structure 7) where the reaction has reached thedead-end product phosphohistidine. The coordination of His170 and His448 by Asp473 and Asp202, respectively, isincluded to illustrate the numerous interactions in this region.


finding is fully supported by the present study onPLDPMF, and implies the possibility of a moregeneralized reaction mechanism throughout thePLD superfamily of enzymes.

Further hydrolysis

The finding that a phosphohistidine could beidentified in the active site of PLDPMF in the firstcrystal structure of substrate soaks (structure 6,eight day soak; Figure 5(a)) came as a surprise.The presence of a covalently attached phosphatewould imply that, at least in vitro, in addition toits already known catalytic ability towards theterminal phosphoester bond of a phospholipidsubstrate (catalytic action on phosphodiester sub-strates), PLDPMF is able to catalyze the hydrolysisof phosphomonoesters. PLDPMF has been said tohave catalytic preference towards a choline head-group, and not towards the fatty acid backbone ofthe substrate phospholipid (S. Servi, personal com-munication). One would therefore not expect thatthe product PA could bind to the enzyme to befurther cleaved into DAG and phosphate. The firststep towards the formation of the two products,choline and PA, is the loss of choline. If the actualend-point of catalysis in crystals is the formationof a phosphorylated histidine, the product PA hasto re-enter the active site in order to make this reac-tion possible. The product PA would then have tore-enter the active site with the DAG (lipid back-bone) moiety in an apical position to His170. Inorder for this to happen, the specificity pocket ofPLDPMF must be large enough to accommodate theDAG moiety of diC4PA. An estimate of the electro-static surface potential in the active site region ofstructure 4 (30 minutes substrate soak) can be seenin Figure 7. For clarity, the diC4PA product hasbeen left out of the surface calculations. From thisFigure, it is clear that the direction of the head

group when the substrate enters the active site istowards the left, and the size of the head group isclearly limited by residues Gln342, Gly389 andTyr390. The latter two are neighbouring residuesto one of the flexible loops surrounding the activesite. In most of the crystal structures presentedresidues 383–388 are missing. These missing resi-dues could potentially be involved in determiningthe substrate specificity of PLDPMF. However, it isclear from these structures that the substrate speci-ficity of PLDPMF is indeed broad. This is consistentwith the general observation that bacterial PLDshave a wider tolerance towards the size of the alco-hols accepted in the transphosphatidylation reac-tion. By analogy to the apical water attackobserved from structure 4 (Figure 3), the nucleo-philic alcohol in the transphosphatidylation reac-tion would have to attack the covalentphosphohistidine intermediate from this apicalposition, consistent with an associative in-line reac-tion. If one considers the charge distributionaround the phosphorus atom in the substrate(phosphodiester) versus the product (phosphomo-noester), the electron withdrawing groups are theoxygen atoms. In a phosphodiester, oxygen atomscan obtain electrons both from the two generalelectron-donating R-groups and from the phos-phorus atom, whereas in a phosphomonoesterthere is only one such R-group. The phosphorusatom in a phosphomonoester is thus likely to be abetter electrophile and thereby more susceptible tonucleophilic attack from the enzyme. When thishypothesis is tested by product soaks (structure 7and 8; Figure 5(b) and (c)), it becomes evidentthat His170 is a sufficiently good nucleophile toform the covalently attached intermediate andhydrolyze the glycerol group, analogous to thefirst step in the substrate binding. However, anactivated water molecule is not a sufficiently goodnucleophile to release the phosphate group fromthe enzyme, and therefore the reaction terminateson the formation of the phosphohistidine. This isalso supportive to the assumption that the rate-lim-iting state for catalysis in crystals of PLDPMF is therelease of the covalent enzyme–product intermedi-ate in the substrate soaks, indicating why it is poss-ible to trap the reaction in flash-cooled crystals.

Reaction mechanism

From the presented results, a general reactionmechanism can be outlined (Figure 8). From thecrystal structures of PLDPMF interacting withshort-chained substrate or product, it is clear thatthe two histidine residues in the active site indeedhave different roles during catalysis, with the N12

side-chain atom of the N-terminal domain His170acting as the nucleophile which attacks the phos-phorus atom of the substrate phospholipid. This isfollowed by the formation of a short-lived five-coordinated phosphate intermediate, before ahydrogen is donated from the C-terminal domainHis448 to produce choline, ethanolamine or serine

Figure 7. Electrostatic surface potential of the activesite of PLDPMF from the 30 minutes substrate soak(structure 4) excluding diC4PA from the calculation. TheFigure was made using the SwissPDBViewer,46 with anelectrostatic surface potential imported from GRASP47

and contoured at ^10 kT/e, where red describes anegative and blue a positive potential.


depending on the substrate. This results in aninversion of the configuration of the phosphorusatom to form a stable tetrahedral reaction inter-mediate with a covalent P–N bond to His170 N12.As His448 donated a proton to the first leavinggroup, this residue is now able to (partially)deprotonate a nearby water molecule (OW in allFigures of the presented crystal structures), either

simultaneously with a nucleophilic attack on thestable covalent PA–histidine intermediate, or in atwo-step process. This results in the formation ofthe second product, PA. Again, the reactionproceeds via the formation of a short-lived, five-coordinated phosphorus intermediate. This resultsin a second inversion of configuration of thephosphorus atom, and thus overall retention of

Figure 8. The reaction mechanism for PLDPMF on a phosphatidylcholine (PC) substrate. R, Diacylglycerol (DAG); R0,choline. The reaction that takes place when the product re-enters the active site and the dead-end phosphohistidine isformed is illustrated below the horizontal line.


configuration in the total reaction. This is thereaction mechanism for a general phospholipidsubstrate.

If one uses an artificial substrate where thelengths of the fatty acid chains at the sn-1 and sn-2positions of the glycerol backbone have beenreduced in order to increase solubility, the productdiC4PA can re-enter the active site to produce aphosphorylated enzyme for a second time, follow-ing the same procedure as already described, i.e.resulting in release of DAG and an inversion ofconfiguration for the phosphorus atom in an in-lineassociative mechanism. However, this time a(partially) deprotonated water molecule is not asufficient nucleophile to release phosphate fromthe enzyme, and a dead-end state is reached. Itmust be emphasized that it is highly unlikely thatPLDPMF would hydrolyze PA in vivo and that thepresent finding is probably an artefact of theexperimental conditions. It did, however, lead to aseries of experiments that have given additionalinformation and greater understanding of thereaction mechanism of this enzyme.

Materials and Methods

Reagents

All compounds used in soaking experiments ortested for solubility were purchased from Sigma.These were 1,2-dibutyryl-sn-glycero-3-phosphoryl-choline (diC4PC), 1,2-dicaproyl-sn-glycero-3-phosphoryl-choline (diC6PC), 1,2-dioctanoyl-sn-glycero-3-phosphate(diC8PA), 1,2-diacyl-sn-glycero-3-phosphorylcholine(phosphatidylcholine; diCnPC), glycerophosphate,glycerophosphorylcholine and sodium tungstate. Otherchemicals were of analytical purity.

Crystallization

Crystallization of PLDPMF was performed asdescribed34 yielding thin plates with dimensions ofabout 0.05 mm £ 0.2 mm £ 0.3 mm. Soaking experimentswere carried out using an excess of the substrate orproduct-compounds (see Table 1 for concentrations). ForX-ray intensity data collection, crystals were cryo-cooleddirectly in the 100–120 K nitrogen gas stream or pre-cooled in liquid N2.

Preparation of complexes

Structure 1: unliganded PLDPMF

Data were collected on crystals backsoaked intothe phosphate-free soaking solution buffer A(0.2 M ammonium acetate, 30% (w/v) PEG4000,buffered with 0.1 M sodium acetate at pH 5.4). Nosign of remaining phosphate in the active sitecould be seen in the electron density maps.

Structure 3: PLD–WO4

Crystals were prepared for the MAD experimentwhich lead to structure solution,15 by backsoakingcrystals in buffer A before transferring the crystals

into a new drop having 10 mM of sodium tung-state added to buffer A. The crystals were soakedfor a period of 48 hours before data were collected.

Structures 4–8: substrate and product soaks

Substrates or products having chain lengths ofsix carbon atoms or longer were generally foundto be insoluble in the mother liquor and 1,2-dibu-tyryl-sn-glycero-3-phosphorylcholine (diC4PC) wastherefore used as substrate. The general approachfor solubilization was that a suitable amount ofthe substrate dissolved in chloroform was trans-ferred with a microsyringe to a glass well and leftuntil the chloroform had evaporated. Buffer A wasthen added to the well and mixed. Crystals weretransferred into this drop and left to soak untildata collection. In the case of glycerophosphate, asample was weighed out and added to a well,before the same procedure as described above wasfollowed. Crystals were tested until satisfactorycooling and diffraction to a reasonable resolutionwere obtained. In cases where the crystals did notcool properly, the PEG-concentration wasincreased by adding a few microliters of 50% ofPEG4000 to the stabilizing solution.

An important advantage, both of the original crystal-lization conditions and the soaking solution, was thatthey both served as a cryo-protecting solution so thatcrystals could be cooled without an intermediate step.

Data collection

X-ray intensity data were collected at three differentbeamlines at the ESRF (the Swiss-Norwegian CRG Beam-line; SNBL, the MAD-dedicated beamline; BM14, and thetunable undulator beamline ID14-EH4). All crystals aremonoclinic, space group P21 with the unit cell par-ameters varying moderately (Table 1), depending oncompound soaking-time and ligand concentration usedin the actual experiment. Data collection was carried outat 100 K or 120 K.

As the data were collected over a relatively longperiod of time and at several beamlines, differentapproaches were applied regarding data collectionstrategies. Most often, the program STRATEGY35 wasused to determine the shortest possible rotation rangeneeded to collect complete data. As shown in Table 1,all data sets are more than 90% complete.

The data were integrated in DENZO36 with scalingand merging of the data sets using SCALA from theCCP4 program suite.37 A total of eight data sets wereused in this comparison. Together with refinementstatistics, these are summarized in Table 1.

Model building and refinement

As described,15 the crystal structure of PLDPMF wasdetermined by applications of the MAD method.38,39

Initial phases gave an electron density map of highquality, leading via solvent flattening and phase exten-sion techniques with DM40 to the refined 1.4 A structureof PLDPMF with PO4 bound in the active site (structure 2in Table 1). After model building in O,41 the proteinmodel was initially refined in X-PLOR42 and thereafterin SHELXL.43 All other presented structures were refined


in REFMAC5,44 using the model of PLDPMF with boundphosphate as the starting model. Before starting refine-ment, the two phosphate groups and all water moleculesin, or near the active site of PLDPMF were removed. Afterrefinement, water molecules with poor fit in electrondensity were removed from the model. Subsequentcycles of refinement and model building brought eachof the individual models to convergence. The refinementprogress was monitored by excluding a fraction of thereflections (typically 2000) from the refinement forcross-validation purposes.45 Manual rebuilding wascarried out using the program O41 with sA-weighted26

2mFo 2 DFc and mFo 2 DFc electron density maps.

Atomic coordinates

The structure factors and structures for structure1 to 8 have been deposited in the Protein DataBank and have accession numbers 1v0s, 1f0i, 1v0r,1v0y, 1v0w, 1v0v, 1v0t and 1v0u, respectively

Acknowledgements

This work was supported by the ResearchCouncil of Norway (I.L. and E.H.). ESRF isacknowledged for financial support for datacollection. All beamline staffs are thanked forexpert technical help. Professor ThorleifAnthonsen and Dr Tore Lejon are acknowledgedfor helpful discussions regarding the reactionmechanism. Professor Stefano Servi and DrFrancesco Secundo are acknowledged for helpfuldiscussions and generous supplies of pure protein.Dr Hanna-Kirsti S. Leiros and Dr GordonA. Leonard are thanked for helpful discussions.

References

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Table 1. Statistics from data collection, processing and refinement

Native Inhibitor Substrate Product

Ligand – PO4 WO4 diC4PC GlycerophosphateStructure

number1 2 3 4 5 6 7 8

Beamline SNBL SNBL BM14 SNBL SNBL ID14-EH4 ID14-EH4 ID14-EH4Detector Image plate

MAR300Image plate

MAR300Image plate

MAR345Image plate

MAR345Image plate

MAR345ADSC-CCD

ADSC-CCD

ADSC-CCD

Temperature(K)

120 110 100 120 120 100 100 100

Soaking time – – Two days 30 minutes Eight hours Eight days 30 minutes 90 minutesConcentration

(mM)– – 10 2 2 2 2 2

Data collectionWavelength (A) 0.873 0.873 0.886 0.873 0.873 0.934 0.934 0.934Unit cell

dimensionsa ¼ 57.41 A a ¼ 57.28 A a ¼ 57.61 A a ¼ 57.37 A a ¼ 57.23 A a ¼ 57.45 A a ¼ 57.16 A a ¼ 57.39 A

b ¼ 57.37 A b ¼ 57.42 A b ¼ 56.90 A b ¼ 56.46 A b ¼ 56.37 A b ¼ 56.75 A b ¼ 56.94 A b ¼ 57.04 Ac ¼ 68.62 A c ¼ 68.7 A c ¼ 68.95 A c ¼ 68.76 A c ¼ 68.63 A c ¼ 68.54 A c ¼ 68.81 A c ¼ 68.76 Ab ¼ 93.458 b ¼ 93.178 b ¼ 93.678 b ¼ 93.748 b ¼ 93.698 b ¼ 93.398 b ¼ 93.488 b ¼ 93.048

Max.diffraction (A)

1.75 1.4 1.7 1.7 1.35 1.7 1.53 1.42

Completeness(%)

92.3 99.2 99.6 98.9 99.0 91.2 95.9 94.9

Redundancy 1.8 3.0 3.8 4.9 4.7 3.3 3.4 3.3Rmerge (%)a 8.0 5.8 4.3 11.5 5.0 6.4 5.1 4.9I/sIb 8.4 (2.0) 7.4 (3.4) 11.8 (2.9) 4.5 (2.0) 8.5 (2.0) 6.5 (4.9) 8.6 (3.0) 9.3 (2.0)

Refinement dataProgram used REFMAC5 SHELXL REFMAC5 REFMAC5 REFMAC5 REFMAC5 REFMAC5 REFMAC5Resolution (A) All data 10–1.4 All data All data All data All data All data All dataRcryst (%) 15.1 12.98 15.5 16.0 16.2 16.3 15.7 16.7Rfree (%) 20.7 18.51 19.8 19.8 18.6 20.9 19.0 18.8Average

B-factor (A2)12.90 13.86 19.09 20.28 13.69 18.27 15.55 15.46

GeometryBonds (A) 0.019 0.0110 0.018 0.019 0.011 0.019 0.014 0.013Angles (deg.) 1.755 2.009 1.999 2.005 1.954 2.019 1.868 2.048ESU (A)c 0.12 (0.12) 0.09 (0.09) 0.10 (0.10) 0.11 (0.10) 0.06 (0.06) 0.12 (0.12) 0.08 (0.08) 0.07 (0.07)

a Rmerge ¼ ðP

h

Pi lIiðhÞ2 kIðhÞllÞ=ð

Ph

Pi IðhÞÞ, where IiðhÞ is the ith measurement of reflection h and kIðhÞl is the weighted mean of

all measurements of h.b The numbers in parenthesis are for the outermost shell (tenth bin) of reflections.c Estimated overall coordinate error. The numbers in parenthesis are for the fraction of reflections kept for cross-validation

purposes.


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Edited by R. Huber

(Received 15 December 2003; received in revised form 25 March 2004; accepted 6 April 2004)


Documents

The Reaction Mechanism of Phospholipase D from Streptomyces sp. Strain PMF. Snapshots along the Reaction Pathway Reveal a Pentacoordinate Reaction Intermediate and an Unexpected Final