doi:10.1016/j.jmb.2008.10.048 J. Mol. Biol. (2008) 384, 1353–1368

Available online at www.sciencedirect.com

Specificity and Reactivity in MenaquinoneBiosynthesis: The Structure of Escherichia coli MenD(2-Succinyl-5-Enolpyruvyl-6-Hydroxy-3-Cyclohexadiene-1-Carboxylate Synthase)

Alice Dawson, Paul K. Fyfe and William N. Hunter⁎

Division of Biological Chemistryand Drug Discovery, College ofLife Sciences, University ofDundee, Dundee, DD1 5EH,UK

Received 20 August 2008;received in revised form13 October 2008;accepted 14 October 2008Available online1 November 2008

*Corresponding author. E-mail addrw.n.hunter@dundee.ac.uk.Abbreviations used: AU, analytica

CEAS, N2-(2-carboxyethyl)arginine scoenzyme A; NCS, noncrystallograpoxalyl-CoA decarboxylase; PDB, Propyruvate oxidase; SAD, single-wavediffraction; SEPHCHC, 2-succinyl-5-hydroxy-3-cyclohexadiene-1-carboxyL-selenomethionine; SHCHC, 2-succcyclohexadiene-1-carboxylate; ThDPdiphosphate.

0022-2836/$ - see front matter © 2008 E

The thiamine diphosphate (ThDP) and metal-ion-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase,or MenD, catalyze the Stetter-like conjugate addition of α-ketoglutarate withisochorismate to release 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexa-diene-1-carboxylate and carbon dioxide. This reaction represents the firstcommitted step for biosynthesis of menaquinone, or vitamin K2, a keycofactor for electron transport in bacteria and a metabolite for posttransla-tional modification of proteins in mammals. The medium-resolutionstructure of MenD from Escherichia coli (EcMenD) in complex with itscofactor and Mn2+ has been determined in two related hexagonal crystalforms. The subunit displays the typical three-domain structure observedfor ThDP-dependent enzymes in which two of the domains bind and forcethe cofactor into a configuration that supports formation of a reactiveylide. The structures reveal a stable dimer-of-dimers association inagreement with gel filtration and analytical ultracentrifugation studiesand confirm the classification of MenD in the pyruvate oxidase family ofThDP-dependent enzymes. The active site, created by contributions from apair of subunits, is highly basic with a pronounced hydrophobic patch.These features, formed by highly conserved amino acids, match well to thechemical properties of the substrates. A model of the covalent intermediateformed after reaction with the first substrate α-ketoglutarate and with thesecond substrate isochorismate positioned to accept nucleophilic attack hasbeen prepared. This, in addition to structural and sequence comparisonswith putative MenD orthologues, provides insight into the specificity andreactivity of MenD and allows a two-stage reaction mechanism to beproposed.

© 2008 Elsevier Ltd. All rights reserved.

Keywords: crystal structure; enzyme mechanism; menaquinone biosynthesis;thiamine diphosphate cofactor

Edited by G. Schulz

ess:

l ultracentrifugation;ynthase; CoA,hic symmetry; OXC,tein Data Bank; POX,length anomalousenolpyruvyl-6-late; SeMet,inyl-6-hydroxy-2,4-, thiamine

lsevier Ltd. All rights reserve

Introduction

Menaquinone, or vitamin K2, is a lipid-solublemolecule with distinct biological functions accord-ing to the type of organism in which it is found.Its chemical properties are exploited in theelectron transport chain in Gram-positive aerobesand facultative Gram-negative bacteria underanaerobic conditions.1,2 In an aerobic environment,Gram-negative organisms utilize ubiquinone.3,4

In mammals, which acquire menaquinone fromintestinal microflora and diet, menaquinone is animportant enzyme cofactor involved in glutamate

d.

1354 Structure Mechanism of MenD

γ-carboxylation.5 This is an important posttransla-tional modification of certain proteins involved inblood coagulation, bone metabolism, and vascularbiology.The biosynthesis of menaquinone has been exten-

sively studied, mainly in Escherichia coli, and shownto involve a pathway of eight or nine proteinsexploiting a range of cofactors.1,2 The starting pointis chorismate, the branch point intermediate of theshikimate pathway.6 Chorismate is isomerized toisochorismate and then converted, in two stages, to o-succinyl-1-benzoate. Two further enzyme-catalyzedreactions, involving ATP and CoA (coenzyme A), ledto a CoA-naphthalene derivative and a thioesteraseremoves the CoA moiety to release 1,4-dihydroxy-2-napthanoate. Prenylation and methylation thenproduce the reduced form of menaquinone. Thestructure–function–mechanism relationships for sev-eral enzymes involved in menaquinone biosynthesishave been elucidated. These include MenB,7–9

MenF,10,11 and MenH.12 A notable exception is theenigmatic enzyme that catalyzes the first committedstep inmenaquinone biosynthesis,MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-car-boxylate synthase).Initially, MenD was assigned as the bifunctional

enzyme 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase1,13 and reactionscatalyzed were considered to be decarboxylation ofα-ketoglutarate and ligation of a second substrate,isochorismate, to produce 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate (SEPHCHC).Cleavage of the pyruvate moiety then producedSHCHC (Fig. 1a). Recently, however, more rigorousanalyses provided evidence that the enzyme isactually SEPHCHC synthase and a complicatingfactor in the characterization of MenD was the slow,spontaneous release of pyruvate from SEPHCHC(Fig. 1a).14,15 The cleavage of pyruvate fromSEPHCHC is catalyzed by MenH (also known asYfbB, Fig. 1a).12 In E. coli, MenH is encoded afterMenD on the same operon as the majority of theother menaquinone biosynthetic genes.The observation that MenD is dependent on the

cofactor thiamine diphosphate (ThDP) and a diva-lent metal cation, with Mg2+ and Mn2+ providingoptimal activity, has structural and mechanisticimplications.13,14 ThDP, the active form of vitaminB1, consists of a diphosphate moiety, aromaticthiazolium, and aminopyrimidine rings. Tautomer-ization of the aminopyrimidine is an importantchemical property of the cofactor (Fig. 1b). It hasbeen demonstrated by spectroscopic methods thatthe N4′-aminopyrimidine and N1′,N4′-iminopyri-midine tautomers are present in a number ofpyruvate decarboxylases with the latter form inparticular present in the predecarboxylationintermediate.16,17 Within the confines of an enzymeactive site, ThDP adopts a bent conformation thatsupports deprotonation of C2 by the basic imine N4′to produce the activated form of the cofactor, anucleophilic ylide (Fig. 1b). The interaction of aglutamate, highly conserved in ThDP-dependent

decarboxylases, with the aminopyrimidine N1′supports the generation of the N4′-iminopyrimidineform that can acquire a proton from the thiazoliumC2. It is this almost universal triumvirate ofglutamate–iminopyrimidine–thiazolium in ThDP-dependent enzymes that determines reactivity atC2 and the generation of the reactive ylide.18–21MenD is the only enzyme known to catalyzeaddition of a ThDP intermediate to the β-carbon of asecond substrate, a reaction that is similar to theStetter reaction, which is a 1,4-addition, or conjugateaddition, of an aldehyde to a β-unsaturatedcompound.22 The Stetter reaction is catalyzed bycyanide or thiazolium salts.A complication in identifying potential active-site

residues is the low level of sequence conservation ofThDP-dependent enzymes.21 Even within MenDsequences from different organisms, the identity istypically 20–30% (data not shown), and structuraldata are critical to delineate the important constitu-ents of the enzyme active site. We set out therefore tocharacterize the structure of MenD and improve ourunderstanding of menaquinone biosynthesis. Crys-tallization of the E. coli enzyme (EcMenD) haspreviously been reported23 and we now present themedium-resolution structure of the EcMenD:ThDP:Mn2+ complex. The structure has been exploited toconstruct a model of the first covalent intermediateformed following reaction with α-ketoglutarate,together with the second substrate isochorismate.This, in combination with the analysis of orthologousMenD sequences and comparisons with relatedenzymes, provides a framework for discussion ofsubstrate recognition, the chemical determinants ofcatalysis, and the enzyme mechanism in the firstcommitted step of menaquinone biosynthesis.

Results and Discussion

Quaternary structure

It was important to characterize the quaternarystructure of EcMenD (556 residues; molecular mass,∼61.4 kDa) since all ThDP-dependent enzymes formdimeric or tetrameric structures, with the cofactorbinding and active sites formed by contributionsfrom at least two subunits. An efficient recombinantprotein expression system for an N-terminal hex-ahistidine-tagged EcMenD was prepared and ahighly purified enzyme was obtained with a yieldof approximately 100 mg L−1 of culture. The finalpurification step employed analytical gel filtrationand indicated that the bulk of the apoprotein,approximately 75%, eluted with a mass of about150 kDa and that 25% eluted with a mass ofapproximately 300 kDa (data not shown) irrespec-tive of whether the histidine tag was present (nativeEcMenD) or had been removed by proteolysis as inthe case of a selenomethionine derivative (SeMetEcMenD). Although these data indicate that a dimeris the predominant form of EcMenD in solution with

Fig. 1. The reactions catalyzed by MenD and MenH and tautomeric forms of the pyrimidine moiety of the cofactor. (a) MenD catalyzes the reaction of isochorismate (5S,6S)-5-[(1-carboxylatoethenyl)oxy]-6-hydroxycyclohexa-1,3-diene-1-carboxylate with α-ketoglutarate to produce SEPHCHC and carbon dioxide. MenH catalyzes the cleavage ofpyruvate from SEPHCHC to release SHCHC. This reaction can also occur spontaneously. M2+ represents Mg2+ or Mn2+. (b) Chemical structure and numbering scheme for ThDP.Three tautomeric forms of the pyrimidine moiety are implicated in ylide generation. PP represents the cofactor diphosphate.

1355Structure

Mechanism

ofMenD

1356 Structure Mechanism of MenD

a smaller quantity of tetramer present, we note thatthe observed mass differs significantly from thetheoretical mass of the dimer (∼123 kDa). The peakcorresponding to the dimer was isolated and thatprotein was used for crystallization and analyticalultracentrifugation (AU). AU was performed onuntagged apo-EcMenD and in the presence of ThDPand Mn2+ (data not shown). The apo-EcMenDsample displayed a single species with a mass of∼117 kDa, which more closely agrees with thetheoretical mass of the dimer than observed by gelfiltration. In the presence of ThDP and a divalentcation, a dimer (∼130 kDa) was still predominantbut there was also evidence for monomer(∼60.8 kDa), trimer (∼190 kDa), and tetramer(∼252 kDa). The data indicate that the mostabundant species of EcMenD is a dimer but thathigher-order assembly into a tetramer is alsoobserved. Under the conditions used for AU, theholoenzyme quaternary structure is not particularlystable, hence the observation of a monomer, thepresence of which allows for interaction with thedimer to provide a trimeric species. The differencesobserved between apo- and holoenzyme suggest thepossibility that ThDP and metal ion binding mayinduce gross conformational changes.

Structure determination

The apoenzyme did not crystallize but holo-EcMenD provided two similar hexagonal forms,both of which diffract to medium resolution. Thenative protein, with the N-terminal His-tag, crystal-lized readily in space group P6522 with approximate

Table 1. Crystallographic statistics for EcMenD

Resolution range (Å)Unit cell dimensions: a, c (Å)Space groupNo. of reflectionsNo. of unique reflectionsRedundancyWilson B (Å2)Completenessa

⟨I/σ(I)⟩RmergeProtein residues (A/B/C/D)Water molecules/Cl−

Rwork/RfreeAverage B-factors per subunit [overall/side chain/main chain] (Å2)ABCDWaters/ThDP/Mn2+/Cl−

Ramachandran plot analysisFavorableAdditionally allowedGenerously allowed

DPIb (Å)a Values in parentheses refer to the highest-resolution shell; 2.88–2.b The diffraction-component precision index as defined by Cruicks

unit cell lengths of a=b=93 Å and c=465 Å. A SeMetderivative was prepared but failed to provide use-able crystals under conditions used for the nativeenzyme. Removal of the histidine tag by proteolyticcleavage and further screening identified differentconditions for crystal growth. These SeMet EcMenDcrystals display space group P65 with approximateunit cell lengths of a=b=96 Å and c=463 Å. Despitesimilar unit cell dimensions, the merging of diffrac-tion data for SeMet EcMenD in point group 622 gavean Rmerge of 32% while native EcMenD data gave anRmerge of 8.5% in this high-symmetry system (Table 1).A self-rotation function for the SeMet EcMenD datain P6 indicated that a noncrystallographic symmetry(NCS) 2-fold axis was displaced by 3.5° from theposition of a crystallographic 2-fold in P622. TheMatthews coefficient25 is estimated as 2.4 Å3/Da,and the bulk solvent content is at approximately 50%for each crystal form assuming that the asymmetricunits of native and SeMet EcMenD contain two andfour subunits, respectively.A single-wavelength anomalous diffraction (SAD)

phasing experiment at the Se K-edge providedphases to 2.8 Å resolution for SeMet EcMenD andan interpretable electron density map. Once twosubunits had been sufficiently modeled, a switch tothe more ordered native structure data at 2.5 Åresolution was made using molecular replacementand the model was refined. Once completed, thenative model was used to determine the SeMetEcMenD structure to investigate any structuraldifferences between dimer and tetramer forms.Analysis of crystallographic statistics and model

geometry usingMolProbity26 indicates that acceptable

SeMet derivative Native

83.0–2.8 92.8–2.595.83, 463.17 93.43, 465.47

P65 P6522598,083 286,46257,944 42,93210.3 6.740.8 44.2

99.5 (96.8) 99.3 (95.4)20.6 (6.4) 18.2 (4.5)9.9 (26.2) 8.5 (28.4)

556/556/556/556 554/556395/14 246/016.9/23.3 21.1/29.3

20.1/20.5/19.7 30.5/30.9/30.120.0/20.5/19.6 29.7/30.2/29.219.9/20.3/19.5 –19.9/20.3/19.5 —

14.6/17.5/17.8/29.6 26.6/22.3/25.1/–

96.16 96.23.47 3.440.36 0.360.35 0.34

8 Å (SeMet), 2.56–2.5 Å (native).hank.24

1357Structure Mechanism of MenD

medium-resolution models have been produced(Table 1). Continuous electron density is observedfor almost the entire polypeptide chains in the SeMetEcMenD structure, missing only the additional N-terminal residues (Gly–His) that remain after proteo-lytic cleavage of the His-tag. The native EcMenD stillcarries the His-tag but there is no electron density forthose residues or for Gly176 and Glu177 of subunit Aof native EcMenD. These are in the loop linking β6and α6, a region of polypeptide that displays highthermal parameters in all subunits. An example ofelectron density is shown when the cofactor isdescribed below.Six independent EcMenD subunit structures have

been determined from the two crystal forms. Theseare highly conserved within each asymmetric unitand between native and SeMet structures. Forexample, the 554 Cα positions of subunits A and Bin native EcMenD overlay with a root-mean-squaredeviation (r.m.s.d.) of 0.36 Å, while subunit A in thenative structure overlays with an r.m.s.d. of 0.32 Åonto subunit A of SeMet EcMenD. Intriguingly, thesimilarity extends to a dimer-of-dimers association.In native EcMenD, a crystallographic 2-fold axis,one-third along c bisecting the a–b plane, generates atetramer. In SeMet EcMenD, this crystallographicaxis is converted to an NCS 2-fold axis and theasymmetric unit is a tetramer. The asymmetric unittetramer of SeMet EcMenD superposes on theasymmetric dimer plus symmetry-related dimer ofnative EcMenD with an r.m.s.d. of 0.53 Å over 2220Cα positions. Given such a high degree of similarity,it is only necessary to detail the more ordered nativestructure. We concentrate on subunit A, and wherecontributions from the partner subunit are impor-tant, the subunit is assigned in residue naming.

The EcMenD subunit and dimer formation

The EcMenD subunit has approximately 65% ofresidues in elements of secondary structure anddisplays the three-domain architecture typical ofThDP-dependent enzymes (Figs. 2 and 3a).28 The N-terminal domain (I) covers approximately 200residues. An ordered segment of extended structurelinks domain I to domain II, which spans residues210–340. A slightly shorter linker region then leadsto the C-terminal domain III. Domains I and IIIdisplay similar topology, each consisting of a centralsix-stranded parallel β-sheet sandwiched betweenseveral α-helices (Figs. 2 and 3a). The principaldifference in the two domains resides in the looplinking β4 and β5 and the equivalent in domain III,the segment linking β17 to β18. The polypeptideadopts a different orientation, displays additionalelements of secondary structure, and is longer indomain III compared to domain I. The smallercentral domain (II) has closely related topology, witha five-stranded parallel β-sheet and six helices. Thesheet gains an extra strand by extension across adimerization interface (discussed below).Structural comparisons between EcMenD and

other ThDP-dependent enzymes were explored

with DALI29 and the Secondary Structure Matchingserver30 and now clearly indicate that, as predicted,MenD belongs in the pyruvate oxidase (POX) familyof ThDP-dependent enzymes, the largest of the fiveThDP-dependent enzyme families.28,30 Monomer Aof native EcMenD overlays with a monomer ofbenzoylformate decarboxylase [Protein Data Bank(PDB) code: 1BFD31]with an r.m.s.d of 2.54Å over 426aligned Cα positions, despite a sequence identity ofonly 16%. Similarly, a monomer of oxalyl-CoAdecarboxylase (OXC; PDB code: 2C3132), whichshares 14% sequence identity, overlays with EcMenDwith an r.m.s.d. of 2.56 Å over 424 aligned Cα

positions. The least-squares fit of individual domainsgives r.m.s.d. values that are less than the overallalignments. For example, overlay of the threedomains of EcMenD with the individual domains ofOXC results in r.m.s.d. values of 2.1 Å (156 Cα

positions), 2.5 Å (94 Cα positions), and 1.9 Å (150 Cα

positions), respectively. This suggests that domains Iand III, which bind the pyrimidine and diphosphatecomponents of the cofactor, respectively, are structu-rally more conserved between EcMenD and POXfamily members than the central domain II. SeveralThDP-dependent enzymes, including POX and acet-ohydroxy acid synthase,33 bind a nucleotide indomain II. This nucleotide can fulfill a purelystructural role, like the flavin adenine dinucleotidein acetohydroxy acid synthase, or alternativelyparticipate in catalysis as exemplified by flavinadenine dinucleotide in POX.34 MenD is not depen-dent on binding a nucleotide in domain II, and theoverlay of EcMenD with POX shows that thenucleotide-binding region of POX is replaced by theloop linking β11 with α11 in EcMenD (data notshown). This loop appears therefore to substitute forthe stabilizing effect a nucleotide has on the structureof a subset of ThDP-dependent enzymes.A comparison of 78 MenD sequences indicates a

low level of sequence conservation for this enzymewith only 46 residues out of 556 being highlyconserved (Fig. 2). The MenD sequence list wasderived by omitting those that displayed a sequenceidentity of ≥90% since they were all from closelyrelated species. The lowest level of conservationoccurs in the central domain II (2 residues), followedby domain III (with 16), and the most conserveddomain is domain I with 28 positions conserved.The EcMenD dimer is formed by interactions

involving domains I and III (Fig. 3b). We estimatethat 42 hydrogen bonds and six salt bridge interac-tions are involved in stabilizing the dimer togetherwith the additional contribution from two ThDPmolecules binding to each of the subunits. Approxi-mately 50% of atoms at the interface are nonpolar,indicating a significant contribution of van derWaals forces to oligomerization. Analysis using thePISA server35 indicates that 15% (3340 Å2) of thesurface area of a monomer (23,200 Å2) is occludedon formation of the dimer.Both crystal structures contain a dimer-of-dimers,

constituting the asymmetric unit of the SeMetprotein (subunits A, B, C, and D) and generated by

Fig. 2. The primary and assigned secondary structure of EcMenD. α-Helices are shown as cylinders and β-strands are shown as arrows, colored according to the domain towhich they have been assigned. Residues with a black background are conserved in 90% of MenD sequences listed in UniProt,27 which have been filtered to remove those with≥90% sequence identity level (78 sequences in total). The ThDP interacting and metal binding residues are indicated by black stars and potential substrate binding residues aredenoted by yellow circles.

1358Structure

Mechanism

ofMenD

Fig. 3. The secondary, tertiary, and dimer structure of EcMenD. (a) A ribbon diagram of the EcMenD subunit showingthe elements of secondary structure. ThDP is depicted in stick representation colored according to atom type: C, black; N,blue; O, red; S, yellow; and P, orange. The N- and C-terminal positions are labeled, as are the elements of secondarystructure using the scheme employed in Fig. 2. A black star marks the position of ThDP C2. (b) Ribbon diagram of thedimer. Subunit A is red and in the same orientation as in (a); subunit B is blue and the position of the dimer twofold (NCS)is marked. The cofactor is depicted as in (a).

1359Structure Mechanism of MenD

a crystallographic 2-fold axis applied to subunits Aand B in the native structure as described earlier.Subunits C and D of the SeMet structure correspondto crystallographic equivalents of subunits A and Bin the native structure, respectively. Residues ondomain II make the most significant contributions to

the formation of the tetramer, which involves ∼8%(1860 Å2) of the surface area of a subunit. This isprimarily due to interaction with one partnersubunit A with D or B with C (1500 Å2) with asmaller area of interaction (360 Å2) formed bysubunit A with C or B with D (Fig. 4). Two

Fig. 4. The EcMenD tetramer. The tetramer is shown asa van der Waals surface with subunits labeled and coloreddifferently. The dimer of subunits A and B is in the sameorientation as in Fig. 3b.

1360 Structure Mechanism of MenD

secondary-structure elements contribute to the inter-face. An interaction between β8 strands forms atwo-stranded antiparallel β-sheet, while β7 ofsubunit A interacts with β13 of subunit D tocontinue the β-sheet of domain II across the tetramerinterface, so that the sheet in subunit A extends intosubunit D (data not shown). This crystallographicdimer-of-dimers is markedly similar to the structureof benzoylformate decarboxylase, which has thesame secondary-structure elements present at thedimer-of-dimers interface and is tetrameric in bothsolution and crystalline states.31

Cofactor binding

The ThDP-binding site in EcMenD, in commonwith all other ThDP-dependent enzymes, is formedby contributions from the two subunits that com-prise the dimer, and each dimer contains two activesites approximately 20 Å apart (Fig. 3b). The cofactoris positioned by hydrogen bond and van der Waalsinteractions with the protein in addition to metal ioncomplexation. The residues important for cofactorbinding are contributed from domains I and III,the most highly conserved domains across MenDsequences (Fig. 2).The diphosphate and associated metal ion are

firmly anchored by a number of interactions, anda conserved motif (Gly441-Asp442-X25-Asn468-Asn469 in EcMenD)20 generally allows identificationof ThDP binding proteins in the absence of any otherdata. Although the Asn–Asn combination is notstrictly conserved across MenD sequences, anasparagine always appears in at least one position(data not shown). Three segments of the polypeptide

(the residues linking α15–β15, β17–α18, and the N-terminal section of α17) create a polar pocket inwhich Mn2+ is bound with octahedral coordination.The ligands are two oxygen atoms from the dipho-sphate, the main-chain carbonyl of Gly471, the side-chain carbonyl of Asn469, the side chain of Asp442,and a water molecule (Fig. 5). The metal ioninteractions with the cofactor diphosphate aresupplemented by nine direct cofactor–proteinhydrogen bonds formed with main-chain amides(Leu392, Asp442, Leu443, Ser444, Gln473, andIle474), a side-chain amide (Asn469), and side-chainhydroxyls (Ser391 and Ser444). Two ordered watermolecules, one of which is a ligand for the metal ion,also provide bridging interactions between thediphosphate and the protein (data not shown). Twoof these residues, Ser444 and Asn469, are not strictlyconserved and interact using the functional groupson their side chains. Ser444 is present in 37 MenDsequences and replaced by threonine in 12 sequences,suggesting that the side-chain interaction with thecofactor is often conserved. In the remaining 29sequences, an alanine occupies this position, perhapsallowing enough room for awatermolecule to satisfythe hydrogen-bonding capacity of the phosphategroup. The Asn469 interaction with Mn2+ is alsolikely to be conserved. Asn469 is conserved in 65sequences, and an aspartate is observed in the other13 MenD sequences. The substitution of asparaginewould result in the loss of the side-chain amideinteraction with the phosphate group.The thiazolium forms few direct interactions with

the protein but rather is anchored in place by theinteractions of the pyrimidine and diphosphategroups. This may allow a small degree of flexibilityof this part of the cofactor.36 Ser391 OG is 2.8 Ådistant from the thiazolium sulfur, suggesting theformation of a hydrogen bond. This serine side chainalso interacts with the diphosphate, separated by2.8 Å, and it is possible that the diphosphate O isprotonated to support a hydrogen-bonding interac-tion. There are van der Waals interactions formed bythe thiazolium with Ile418 and Phe475 (not shown)located on either side of the inverted V formedbetween the pyrimidine and thiazolium rings and,at the side, Ile474. Hydrophobic residues are alwaysfound in these positions in the cofactor-bindingpocket of ThDP-dependent enzymes. Ile418 formsvan der Waals interactions also with the pyrimidinecomponent and is strictly conserved in MenDsequences. In addition, the thiazolium methylgroup interacts with Pro30 of the partner subunit(data not shown).The pyrimidine moiety is tucked into a cleft

formed by the turn between β15–α16 of subunit Aand the N-terminal sections of α3 and α4 of subunitB. The three functional groups participate in hydro-gen-bonding interactions with subunit B. N1′appears to form a bifurcated hydrogen bond withthe carboxylate of Glu55. N3′ accepts a hydrogenbond donated from the main-chain amide of Ile418,and N4′ donates a hydrogen bond to the main-chaincarbonyl of Ser416. Ile418 of subunit A is on one side

Fig. 5. The cofactor-binding site. The cofactor is shown as in Fig. 3, and C atoms of the protein residues are coloredgreen. Mn2+ and a water molecule are shown as purple and red spheres, respectively. Hydrogen bonds are depicted asblack broken lines, and interactions between Mn2+ and O ligands are shown as purple lines. In the view selected, theinteraction between the cation and Gly471 is obscured. The difference density omit map (black chicken wire) for ThDPcalculated with (Fo−Fc) and αc coefficients and contoured at the 3 σ level. Fo represents the observed structure factors, Fcdenotes the calculated structure factors, and αc indicates the phases calculated without any contributions from ThDPatoms.

1361Structure Mechanism of MenD

of the pyrimidine, as described above, and on theother side is a polar segment where an orderedwater molecule fills the gap between this part of thecofactor and the protein (data not shown).Glu55 is strictly conserved in MenD sequences,

and mutagenesis of this residue in EcMenD abolishesenzyme activity.13 Glu55, together with Asp54 andArg56, forms a conserved motif in MenD. A networkof hydrogen bonds and salt bridges formed byAsp54 and Arg56 with the equivalent residues in thepartner subunit and the presence of Tyr447 (con-served as Tyr or His inMenD) form a tight turn at thestart of α3 and, together with the bulk of thearomatic side chain, make contributions to theassembly of the MenD dimer and serve to positionGlu55 to bind the cofactor and thereby influence thetautomeric state of the pyrimidine (Figs. 5 and 6).This group of residues is worthy of note with respectto comparisons of ThDP-dependent enzymes.ThDP-dependent enzymes are oligomeric, and in-

vestigations into aspects of communication betweenactive sites have been reported.21,37 In the Bacillusstearothermophilus (now Geobacillus stearothermophilus)E1 subunit of pyruvate dehydrogenase, a heavilyhydrated cavity spanning some 20 Å between thetwo ThDP molecules in the dimer was observed.This channel is lined with acidic residues (fouraspartates and six glutamates), and it has beenpostulated that this channel and the associatedwater molecules may assist the shuttling of protonsback and forth through this tunnel, allowing thecofactors to act in reciprocal fashion as general

acids/bases in catalysis, and that this explained therequirement for oligomers, conformational asym-metry, alternating site reactivity, and the kineticproperties of E1 and other thiamine-dependentenzymes.37 While such a theory may apply tosome, it is unlikely to be a universal feature of allThDP-dependent enzymes since the channel isabsent in transketolase,18 OXC,32 and N2-(2-carbox-yethyl)arginine synthase (CEAS)38 and not allmembers of the family display alternating sitereactivity. There is also no channel linking theactive sites in EcMenD where the Asp54–Glu55–Arg56 motif and Tyr447 block access between thetwo active sites (Fig. 6).A key feature of ThDP-dependent enzymes is the

V-shaped conformation forced upon the cofactor byits binding site, with either end tethered to differentdomains (Figs. 5 and 7). The net result is the closeproximity of the pyrimidine N4′ and thiazolium C2.In crystal structures of ThDP itself, the distance is3.9 Å.40,41 In EcMenD, the average distance betweenN4′ and C2, from the six independent subunitstructures, is 3.1 Å. This distance agrees well withother ThDP-dependent enzymes with the exceptionof CEAS where the separation is much shorter ataround 2.3 Å. The proximity of these two atoms, inconjunction with the presence of a glutamic acid–N1′ interaction, pyrimidine tautomerization, andthe positively charged N3 of the thiazolium, allowsabstraction of the acidic C2 hydrogen by N4′ toproduce the activated ylide ready to react withsubstrate.

Fig. 6. A network of interactions at the dimer interface between two cofactor-binding sites. A similar color scheme toFig. 5 is used with the addition that C atoms of residues in subunit B are gray.

Fig. 7. A stereoview of the model for substrates binding to EcMenD. The post-decarboxylation covalent adduct ofThDP and α-ketoglutarate (ThDP⁎) is shown, and the reactive C atoms of this intermediate and isochorismate are 1.6 Åapart. This separation is indicated by a magenta broken line. The polypeptide main chain is shown as a gray ribbon andatomic positions are colored as in the previous figures except that the C atoms of the second substrate, isochorismate, areyellow. A calculated electrostatic surface potential (blue, positive; red, negative; white, neutral) showing the active-sitecleft. The figure was produced using PyMOL1 with Adaptive Poisson–Boltzmann Solver39 with electrostatic potentialisocontours set at +5 kT/e (blue) and −5 kT/e (red).

1362 Structure Mechanism of MenD

1363Structure Mechanism of MenD

With one notable exception, ThDP-dependentenzymes characterized thus far have the equivalentof Glu55 interacting with N1′, an interaction that isvital to activate the cofactor and support formationof the covalent intermediate with the first substrate.The exception is E. coli glyoxylate carboligase wherea valine occupies the position near to N1′.42 Thisenzyme appears to compensate for the lack of a polarinteraction with the pyrimidine moiety with analiphatic, nonpolarizable environment for the thiazolgroup. Such an environment stabilizes the neutralylide 4′-aminopyrimidine form of the cofactor butreduces the reactivity at C2. The reduction inreactivity is likely balanced by the enhanced reac-tivity of the substrate in this case, an aldehyde asopposed to the ketone substrates processed by themajority of ThDP-dependent enzymes. The presenceof a carboxylate to interact with N1′ acceleratesdeprotonation at C2 and in so doing prevents theenzyme getting stuck in a low-energy intermediate.42

A model for substrate (α-ketoglutarate andisochorismate) binding

Only a limited number of ThDP-dependentenzyme substrate complexes have been experimen-tally determined due to the transient nature of theintermediates formed by the cofactor–substratecomplexes.32,43 An elegant study of OXC, usingmodified ThDP derivatives, provided snapshots ofseveral stages in the decarboxylation cycle (OXC hasno ligase activity) and revealed that the covalentThDP–substrate intermediate has an internal hydro-gen bond between the hydroxyl group, remainingafter decarboxylation, and the pyrimidine N4′.32This study provided a template to assist ourmodeling of the first covalent intermediate inEcMenD. The second substrate, isochorismate, waspositioned such that the reactive carbon was 1.5 Ådistant from the nucleophilic carbon of the activatedintermediate and oriented to minimize steric clashwith the protein (Fig. 7). It was clear that conforma-tional changes involving residues in the β1–α2 turnand several side chains were likely to be required forbinding of the second substrate in a productivefashion. However, no attempts were made to gen-erate an energy-minimized structure of the complexsince we preferred to use the model to simply inferregions of the active site that might be relevant tospecificity and reactivity.The EcMenD active site is, as described, formed by

contributions from two subunits. It consists of anarrow cavity having a width and length ofapproximately 8 and 15 Å, respectively, and thedistance from the surface of the protein to ThDP C2is nearly 20 Å (Fig. 7). The wall of the cavity isprimarily basic due to the presence of five arginineresidues (Arg33A, Arg107A, Arg395A, Arg293B,and Arg413B) together with a lysine (Lys292B). Ahydrophobic patch on one side of the active site iscreated by the side chains of residues Ile474B,Phe475B, and Leu478B. These residue types matchwell to the acidic substrates and, in the case of

isochorismate, a significant hydrophobic character.Seven of these nine amino acids are strictlyconserved in MenD sequences (Fig. 2). The twoexceptions, Lys292 and Arg293, are more distantfrom the catalytic center than the other residuesdiscussed (Fig. 7) and are located in domain II,which contains only two highly conserved residues.Lys292 is conserved or replaced by arginine in morethan 90% of MenD sequences, and the side chain ofthis residue could reach over to interact with thesecond substrate depending upon which rotamer ispresent. Arg293 is not well conserved and unlikelyto bind the substrates directly, but in EcMenD, itmay simply contribute to the basic character of theactive site.The model predicts that the nonpolar component

of isochorismate is placed to interact with thehydrophobic patch formed by the strictly conservedresidues, Ile474B, Phe475B, and Leu478B, on oneside of the active site. This isoleucine/phenylalaninepair is important for binding the cofactor asdescribed earlier. The four conserved arginineresidues could potentially interact with three car-boxylate and one hydroxyl group of the substrateshelping to align the reagents for catalysis. Thesearginine side chains all participate in hydrogen-bonding interactions that serve to position them inthe active site (data not shown). Arg413 lies in theloop linking β15 and α16 and is held in place byforming a hydrogen bond to Asn390, a residuemoderately conserved as asparagine or serine inMenD sequences. Arg395 is located towards the endof α15 and interacts with the main-chain carbonyl ofSer392. We also note that an ordered chloride wasobserved in the SeMet EcMenD active site bound tothe side chain of Arg395 (data not shown). In ourmodel, the carboxylate group of the covalentintermediate occupies this anion recognition site.Arg107 is in the loop section following β4 andinteracts with the main-chain carbonyl of Asn117while Arg33 associates with the main-chain carbo-nyl of Phe170 and forms a salt bridge with Glu172.The model suggests that there are no obvious

residues that could be involved in cleaving thepyruvate side chain of the isochorismate derivative,an observation that is consistent with the assign-ment of MenD as SEPHCHC and not as SHCHCsynthase.14

Mechanistic considerations

Our current knowledge of ThDP-dependentenzymes in conjunction with the structural datanow presented supports the mechanism for EcMenDdepicted in Fig. 8. The mechanism is described intwo stages corresponding to the reactions with eachof the two substrates. In similar fashion to CEAS,38

we note the absence of a suitable residue to functionas a general acid/base and the cofactor N4′ istherefore a critical component of the mechanism.At the onset of stage I, the Glu55 ThDP N1′

interaction supports tautomerization of 1′,4′-imino-pyrimidine to the 4′-aminopyrimidine form and

Fig. 8. A two-stage mechanism for catalysis by EcMenD. An asterisk marks the isochorismate C2, which is attacked by the carbanion intermediate.

1364Structure

Mechanism

ofMenD

1365Structure Mechanism of MenD

promotes increased basic character of N4′ (Figs. 1aand 8). This aids acquisition of the acidic protondonated from the thiazolium C2 to generate acarbanion ylide. The cationic thiazolium N3 helps tostabilize this nucleophile. Attack on α-ketoglutarateC2 occurs to generate a covalent ThDP intermediateandN4′ reverts to the imino-form following donationof a proton that converts the α-ketoglutarate O5carbonyl to a hydroxyl group.Decarboxylation occursto produce an enamine and resonance leads toformation of an activated carbanion intermediate.In stage II, the second substrate binds and the

intermediate carbanion attacks isochorismate C2.The ThDP 4′-imino group can abstract a proton fromthe intermediate hydroxyl and revert to the amino-form and in so doing a carbonyl group is formed.C–C bond cleavage and the release of SEPHCHCcomplete the reaction. In common with the obser-vation above concerning activation of the cofactor,we do not identify a likely proton-donating residuein the vicinity of isochorismate C1–C2. While weinvoke the cofactor itself in the activation stage, forstage II of the reaction, it is possible that a watermolecule participates here in the Stetter-like addi-tion. Further data would be required to address thishypothesis.

Materials and Methods

Reagents, preparation of recombinant expressionsystems, and protein purification

Chemicals, of the highest quality available, were sourcedfrom Sigma-Aldrich and VWR International except wherestated otherwise. The menD gene was amplified by PCRfrom E. coli (K12) genomic DNA using 5′-CATATGT-CAGTCAGCGCATTTAACC-3′ and 5′-GGATCCTCA-TAAATGGCTTACCTGC-3′ as the forward and reverseprimers, respectively (Thermo Scientific). These oligonu-cleotides include 5′ NdeI and 3′ BamHI restriction sitesrespectively, indicated in bold. The PCR product wasligated into TOPO-BLUNT-II (Invitrogen) and then sub-cloned into a modified pET15b vector (Novagen), whichproduces a histidine-tagged protein with a tobacco etchvirus protease site. The native and SeMet-labeled proteinswere obtained using the methionine auxotrophic strain E.coli B834 (DE3) (Stratagene). Cells were grown in 1 L ofLuria–Bertani media supplemented with 50 μg mL−1 ofcarbenicillin for production of native protein, while for theSeMet protein, bacteria were cultured in minimal media(Molecular Dimensions) supplemented with SeMet follow-ing an established protocol.44 Gene expression was inducedat 20 °C using 0.5 mM isopropyl-β-D-thiogalactopyrano-side, and growth continued for 16 h at room temperature.Cells were harvested by centrifugation for 25 min at

40,000g at 4 °C, resuspended in lysis buffer (50 mM Tris–HCl, pH7.5, 250 mM NaCl, and 20 mM imidazole)containing DNase I (0.1 mg) and a single tablet of acocktail of ethylenediaminetetraacetic-acid-free proteaseinhibitors (Roche), and lysed using a French press at1000 psi. Insoluble debris was separated by centrifugationat 39,000g for 25 min at 4 °C, and the soluble fraction wasloaded onto a 5-mL HiTrap Chelating HP column (GEHealthcare) precharged with Ni2+. A linear concentrationgradient was applied to the column and EcMenD was

eluted at 170 mM imidazole. Fractions were analyzedusing sodium dodecyl sulfate polyacrylamide gel electro-phoresis and those containing EcMenD were pooled. Thenative protein, still carrying the affinity tag, was furtherpurified using a Superdex 200 26/60 size-exclusion column(GE Healthcare) equilibrated with 50 mM Tris–HCl and250 mM NaCl, pH7.5. Selected fractions were pooled anddialyzed into 20 mM Tris–HCl, 200 mM NaCl, and 10%glycerol, pH8, and concentrated using Amicon Ultradevices (Millipore) to 5 mg mL−1. The protein concentra-tion was determined spectrophotometrically using atheoretical extinction coefficient of 108,775 M−1 cm−1 at280 nm calculated using ProtParam.45 Purification of theSeMet protein involved two additional steps; after elutionfrom the HiTrap column, the hexahistidine tag was cleavedby incubation with a histidine-tagged tobacco etch virusprotease for 3 h at 30 °C followed by dialysis overnight at4 °C to remove imidazole. The sample was subjected to asecond affinity chromatography step, and the flowthrough, containing untagged SeMet EcMenD, was col-lected and applied to the Superdex column as described forthe native protein. The full incorporation of SeMet and thehigh level of sample purity for both samples were verifiedby matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (data not shown). Theyield of SeMet EcMenD (∼35 mg L−1 of culture) was lessthan that obtained for the native protein, ∼100 mg L−1 ofculture. This is likely due to the reduction in bacterial cellmass observed under conditions required for SeMetincorporation.

Analytical gel filtration and ultracentrifugation

Analytical gel filtration experiments were conducted ona Superdex 200 26/60 column calibrated with molecularmass standards: blue dextran (N2000 kDa), thyroglobulin(669 kDa), ferritin (440 kDa), aldolase (158 kDa), con-albumin (75 kDa), ovalbumin (43 kDa), carbonic anhy-drase (29.5 kDa), ribonuclease A (13.7 kDa), and aprotinin(6.5 kDa) (GE Healthcare; data not shown). Sedimentationvelocity experiments were performed (wavelength of280 nm, rotor AN50-TI at 45,000 rpm and 20 °C), using aBeckman Coulter XL-1 analytical ultracentrifuge. Bothnative (tagged) and SeMet (untagged) EcMenD were runin 50 mM Tris–HCl, pH7.5, and 250 mM NaCl atconcentrations of 0.25, 1.0, and 3.0 mg mL−1 in thepresence of ThDP and MnCl2 at concentrations of 0.5 and2 mM, respectively. Samples were centrifuged simulta-neously, and A280 measurements were taken at 5-minintervals for 16 h. The resultant data were analyzed usingthe programs SEDFIT and SEDNTERP.46,47

Crystal growth, structure determination, and refinement

Crystallization

Sitting and hanging drop vapor diffusion methods wereused for screening and optimization, respectively. Theapoenzyme did not crystallize. Holoenzyme was preparedby incubation with 2 mM MnCl2 and 0.5 mM ThDP, andcrystals were obtained using a reservoir (500 μL) compris-ing 100 mM sodium citrate, pH5.6, 8% isopropanol, and14% polyethylene glycol 6K for the native protein and100 mM Tris–HCl, pH8.5, 22% polyethylene glycol 8K,15% glycerol, and 200 mM NaCl for the SeMet derivative.In both cases, the best crystals were obtained from dropsconsisting of 1 μL protein and 2 μL of reservoir. Crystals

†http://pymol.sourceforge.net

1366 Structure Mechanism of MenD

with hexagonal rod morphology appeared after 1–2 daysat room temperature. The maximum dimensions ofnative crystals were 0.25 mm×0.1 mm×0.1 mm, andthe SeMet derivative crystals were much smaller at0.05 mm×0.02 mm×0.02 mm.

X-ray data collection and processing

Crystals were flash cooled by plunging them into liquidnitrogen, mounted on a goniostat, and maintained at−173 °C in a flow of cooled nitrogen. Diffraction proper-ties were characterized with a Rigaku Micromax 007rotating anode R-AXIS IV+ image plate system. The bestsamples were stored for use at the European SynchrotronRadiation Facility (Grenoble, France). Native data werecollected on beamline ID29, and SAD data were measuredon beamline BM14, both with ADSC Q315 CCD detectors.The SAD data were collected at the Se absorption edgedetermined using X-ray Absorption Near Edge Structurespectroscopy at a wavelength of 0.97872 Å. Data proces-sing and scaling were performed using XDS48 andSCALA,49 and statistics are presented in Table 1. Initialattempts to record native data at a synchrotron sourcewere compromised by severe radiation damage; inaddition, both crystal forms displayed a large unit cell cdimension (N460 Å) with mosaic spread estimated at 0.6°.We had to balance the resolution to which we couldobserve diffracted intensities with being able to accuratelyrecord complete and highly redundant data sets withminimum loss of data due to reflection overlaps orradiation damage. The resulting data sets are thereforeartificially truncated, and this is evident from the high⟨I/σ(I)⟩ and low Rmerge values in the outer shells (Table 1).The high level of redundancy will, however, have con-tributed in a positive way to the ⟨I/σ(I)⟩ values.

Structure solution and refinement

Initial phases were obtained by SAD phasing using thepositions of 23 selenium atoms (out of a possible 32) thatwere identified using the program SOLVE.50 The initialfigure of merit was 0.34 for data to 2.8 Å and the Z-scorewas 56.4. An interpretable electron density map wasobtained only for space group P65. Density modificationusing RESOLVE,51 with an estimated solvent content of50%, improved the figure of merit to 0.77. These phasesproduced an electron density map (calculated using theCCP4 suite of programs52) that was readily interpreted(Fig. 4). Automated interpretation of the map withBuccaneer53 identified a few segments of secondarystructure and provided a starting point for model buildingin Coot.54 Two subunits, of the four that comprise theasymmetric unit, were modeled at which point the higher-resolution native data set, with only a dimer in theasymmetric unit, became available. A monomer was usedfor molecular replacement into the 2.5-Å data usingMOLREP.55 Two monomers were positioned giving anR-factor of 43% and a score of 0.58, with the secondhighest solution giving a score of 0.21. This model wassubsequently refined using REFMAC556 with NCSrestraints applied during the early stages of the refine-ment, subsequently relaxed as the refinement progressedand then removed altogether. Refinement, includingisotropic B-factors, was combined with manual rebuildingin Coot and the addition of water molecules and ligands.The identification of Mn2+ positions was confirmed bycomparison of peak heights in omit difference densitymaps, with those observed on S of ordered methionineresidues.

For the sake of completeness and to investigate possibledifferences in quaternary structure, we then used therefined native model to solve the SeMet EcMenDstructure. Molecular replacement calculations positionedfour molecules, with four clear peaks in the rotationfunction, and the refinement was completed with similarprotocols to those adopted for the native structure. Modelvalidation was carried out using MolProbity26 and Coot.The refinement statistics for both of these models are

summarized in Table 1. It has been our general experiencethat for medium- to high-resolution macromolecularstructures, the Wilson plot B-value is similar to the overallisotropic B-factors displayed by the model. In thestructures reported here, we judge that the artificialtruncation of data, described above, is a likely explanationfor the differences (about 20 and 15 Å2 for the SeMet andnative forms, respectively) in these two terms.PRODRG57 was used to construct models and libraries

for both the ThDP–oxoglutarate intermediate and iso-chorismate, and modeling of the EcMenD substratecomplexes was carried out in Coot. Structural super-positions were carried out with LSQKAB.58 Figures wereprepared with Chemdraw (Adept Scientific), PyMOL†,and ALINE (C. S. Bond, personal communication).

PDB accession numbers

Coordinates and structure factors have been depositedwith accession codes 2jla and 2jlc.

Acknowledgements

This study was supported by awards from theBiotechnology and Biological Sciences ResearchCouncil (Structural Proteomics of Rational Targets,BBS/B/14434) and The Wellcome Trust (grantnumbers 082596 and 083481). We thank MarkAgacan for AU measurements and the EuropeanSynchrotron Radiation Facility for beam time andexcellent staff support.

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