J. Biol. Chem.-2008-Fulton-27881-90

Crystal Structure of a UDP-glucose-specificGlycosyltransferase from a Mycobacterium Species*□S

Received for publication, March 6, 2008, and in revised form, July 23, 2008 Published, JBC Papers in Press, July 30, 2008, DOI 10.1074/jbc.M801853200

Zara Fulton‡§, Adrian McAlister‡, Matthew C. J. Wilce‡, Rajini Brammananth§¶, Leyla Zaker-Tabrizi‡§,Matthew A. Perugini�, Stephen P. Bottomley**, Ross L. Coppel§¶, Paul K. Crellin§¶, Jamie Rossjohn‡§1,and Travis Beddoe‡§2

From the ‡Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences,Monash University, Clayton, Victoria 3800, the §Australian Research Council Centre of Excellence in Structural and FunctionalMicrobial Genomics, Monash University, Clayton, Victoria 3800, the ¶Department of Microbiology, School of Biomedical Sciences,Monash University, Clayton, Victoria 3800, the �Bio21 Molecular Science and Biotechnology Institute, University of Melbourne,Parkville, Victoria 3010, and the **Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, MonashUniversity, Clayton, Victoria 3800, Australia

Glycosyltransferases (GTs) are a large and ubiquitous familyof enzymes that specifically transfer sugarmoieties to a range ofsubstrates. Mycobacterium tuberculosis contains a large num-ber of GTs, many of which are implicated in cell wall synthesis,yet themajority of theseGTs remainpoorly characterized.Here,we report the high resolution crystal structures of an essentialGT (MAP2569c) fromMycobacterium avium subsp. paratuber-culosis (a close homologue ofRv1208 fromM. tuberculosis) in itsapo- and ligand-bound forms. The structure adopted the GT-Afold and possessed the characteristic DXD motif that coordi-nated anMn2� ion. Atypical of most GTs characterized to date,MAP2569c exhibited specificity toward the donor substrate,UDP-glucose. The structure of this ligated complex revealed aninduced fit binding mechanism and provided a basis for thisunique specificity. Collectively, the structural features sug-gested that MAP2569c may adopt a “retaining” enzymaticmechanism, which has implications for the classification ofother GTs in this large superfamily.

Mycobacterium tuberculosis, the causative agent of tuber-culosis, is a devastating bacterial pathogen, and infection byM. tuberculosis continues to be common particularly indeveloping countries, with particular strains becomingresistant tomultiple frontline drugs. The pathogenicity ofM.tuberculosis is partly attributable to its waxy cell wall that

consists of covalently linked layers of peptidoglycan, arabi-nogalactan, and mycolic acids (for a review, see Crick et al.(1)), interspersed with lipids and glycolipids, and cappedwith polysaccharides (2). Despite the major importance tothe biology of Mycobacterium sp. and other Corynebacteri-neae, the biosynthetic pathways for many of these carbohy-drates are poorly understood, although it is established thatthe most prolific class of enzymes involved in these pathwaysare the glycosyltransferases (GTs)3 (3). Indeed, there are 41known or putative GTs inM. tuberculosis strain H37Rv listedin the CAZy (carbohydrate-active enzyme) data base, repre-senting over 1% of the 3900 open reading frames in the M.tuberculosis H37Rv genome (4). Ethambutol, a front lineanti-tuberculosis drug, targets the EmbA and EmbB GTsthat synthesize the arabinogalactan cell wall layer, therebymarking GTs as promising new drug targets.GTs transfer sugarmoieties from the activated donor substrate,

mostly in the formofanucleoside-diphospho-sugar, toamyriadofspecific acceptor substrates to generate innumerable oligosaccha-ride and glycoconjugate products that are often required for spe-cies- or cell specific processes. GTs are classified as either “invert-ing” or “retaining,” depending on whether the stereochemistry ofthe anomeric carbon is retained or inverted in the product relativeto that in the donor substrate. Moreover, despite the sequencediversity within the GT superfamily, GT structures solved to dateadopt one of two common folds, termed “GT-A” or “GT-B.” Bothof these folds adopt the “Rossmann-like fold,” with the GT-A foldcontaining a single Rossman-like or nucleotide-binding domain(5) and the GT-B fold containing two similar Rossmann-likedomains. Moreover, members within a particular GT family arepredicted to share the same inverting or retainingmechanismandGT-A or GT-B fold. Furthermore, GTs of the GT-A fold that uti-lize a nucleoside-diphospho-sugar typically contain a “DXD” (orXDD or EXD) motif that is involved in metal ion-mediated acti-vated donor substrate coordination and is also required for cata-lytic activity (6).In contrast to the abundance of GT sequences available,

there is a lack of functional, mechanistic, and structural data on

* This work was supported by the Australian Research Council (ARC) Centre ofExcellence in Structural and Functional Microbial Genomics and theNational Health and Medical Research Council (NHMRC) of Australia. Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1 and Figs. 1– 4.

The atomic coordinates and structure factors (codes 3CKJ, 3CKN, 3CKQ, 3CKV,and 3CKO) have been deposited in the Protein Data Bank, Research Collabo-ratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ(http://www.rcsb.org/).

1 An ARC Federation Fellow. To whom correspondence may be addressed.Tel.: 613-9905-3736; Fax: 613-9905-4699; E-mail: [email protected].

2 A NHMRC Career Development Award Fellow. To whom correspondencemay be addressed. Tel.: 613-9905-3736; Fax: 613-9905-4699; E-mail:[email protected].

3 The abbreviations used are: GT, glycosyltransferase; MGS, mannosylglycer-ate synthase; MAD, multiple anomalous dispersion.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 41, pp. 27881–27890, October 10, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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these enzymes. This paucity of biochemical data reflects thedifficulty in characterizing enzymes with countless possiblecombinations of donor and acceptor substrates and products.Indeed, only 29 of the 90GT families have representative struc-tures solved. The vast majority of sequences uncovered bygenomic analyses that show distant sequence identity to knownGTs are thus grouped into large “polyspecific” families. Forexample, the rapidly growing GT2 family is the largest suchfamily, with over 10,000 known and candidate members, and isconsidered the ancestral inverting family from which all GTsthat share the GT-A fold have evolved (7). The spore coatpolysaccharide biosynthesis protein (SpsA) from Bacillussubtilis (8), which adopts the GT-A fold, remains the onlyrepresentative of the GT2 family whose structure is solved.Due to the versatility of the GT-A fold, “rules” for discerningthe substrate specificity and discriminating the mechanismof GTs that exhibit this fold remain elusive. To further ourunderstanding of this family of GTs and to characterize newanti-tuberculosis drug targets, we investigated Rv1208, anessential enzyme (9) implicated in the biosynthesis of theoligosaccharide- and glycoconjugate-rich M. tuberculosiscell wall. As a candidate member of the GT2 family, Rv1208 ispredicted to use an inverting mechanism, Mn2� and a nucleo-side-diphospho-sugar as its activated donor substrate.We havedetermined the crystal structure of MAP2569c fromMycobac-terium avium subsp. paratuberculosis, a close homologue (83%sequence identity) to Rv1208. We reveal that MAP2569c pos-sesses the GT-A fold and exhibits specificity toward the donorsubstrate, UDP-glucose.

EXPERIMENTAL PROCEDURES

Production and Crystallization of MAP2569c—Detailedmethods used for the cloning, overexpression, purification, andcrystallization of MAP2569c have been published previously(10). Briefly, selenomethionine-labeled protein was overex-pressed in Escherichia coli B834 (DE3) grown in autoinducingmedium, as described by Studier (11) at 25 °C for 24 h. Thecrystallized form of MAP2569c in this study comprised resi-dues 5–329 of the native sequence. MAP2569c crystallized inspace group P41212 with unit cell dimensions a � b � 86.6 Åand c� 104.3 Å. There is onemonomer in the asymmetric unitcorresponding to a solvent content of �54% (v/v).The co-complex crystals were obtained by soaking the

“native” crystals in 1M ammonium sulfate, 0.1MHEPES, pH7.0,or 0.1 M sodium citrate, pH 5.5, 20 mM manganese chloride, 1mM sodium thiosulfate, 5% (v/v) glycerol, and 100 mM UDP orUDP-sugar for 6 h. The crystals were prepared for x-ray datacollection by the addition of 15% (v/v) glycerol prior to flashcooling directly in liquid nitrogen.X-ray Diffraction Data Collection—X-ray diffraction data were

collected from crystals at 100 K on IMCAbeamline 1710-D at theAdvanced Photon Source, Argonne National Laboratory (Chi-cago, IL), using a MAR CCD 165 detector, at General MedicineandCancer InstitutesCollaborativeAccessTeam (GM/CA-CAT)beamline 23 ID-D at the Advanced Photon Source using a MARCCD300detector, and in house using aRigakuRU-H3RHB rotat-inganodegeneratorandR-Axis IV��detector (seeTable1).X-ray

data were processed and analyzed using the CCP4 program suite(12) (see Table 1).Phase Determination,Model Building, and Refinement—The

multiple anomalous dispersion (MAD) technique was used toobtain the initial phases from selenomethionine-substitutedcrystals of apo-MAP2569c. Three selenium atom sites werelocated, and initial phases were calculated using BnP (13). Thefigure of merit was 0.585 to 2.3 Å (see Table 1). The initialphases were extended to 1.8 Å resolution with automated den-sity modification using DM (14). The starting model was builtinto the density-modified electron density map using ARP/wARP (15). The final model was obtained after iterative cyclesof manual model building with COOT (16) and TLS andrestrained refinement using REFMAC (14, 17). The final modelconsists of two contiguous polypeptide chains and comprised300 residues (residues 15–173 and 189–329 of the nativesequence) and 102 water molecules. The structure was refinedto 1.85 Å to Rfactor and Rfree values of 18.4 and 19.8%, respec-tively (see Table 2).For the co-complexation studies, difference Fourier analyses

were used to evaluate nucleotide and nucleotide-sugar binding.Accordingly, unbiased features in the electron density mapsrevealed the location of the nucleotide or nucleotide-sugarmolecules, and the subsequent structures were refined usingsimilar protocols to those listed above (see Tables 1 and 2). Forrefinement of the ligated MAP2569c complexes, the same Rfreedata set was used as selected in the apo crystal form.Analytical Ultracentrifugation Analysis—Sedimentation

velocity experiments were conducted in a Beckmanmodel XL-Ianalytical ultracentrifuge at a temperature of 20 °C. A sample ofMAP2569c (380 �l, 1.5 mg/ml) solubilized in 20 mM Tris, 150mMNaCl, pH 8.0, and reference (400-�l) solutions were loadedinto a conventional double sector quartz cell and mounted in aBeckman four-hole An-60 Ti rotor. Data were collected in con-tinuous mode at 295 nm using a rotor speed of 40,000 rpm, atime interval of 300 s, and a step size of 0.003 cm without aver-aging. Solvent density (1.005 g/ml at 20 °C) and viscosity (1.021cp) as well as estimates of the partial specific volume ofMAP2569c (0.74 ml/g) were computed using the programSEDNTERP (18). Sedimentation velocity data at multiple timepoints were fitted to a single discrete species or a continuoussize distribution model (19) using the program SEDFIT, whichis available on the World Wide Web.Enzyme Activity—The nucleotide-sugar specificity of

MAP2569c was determined by a continuous linked enzymeassay described by Grosselin et al. (20). The 200-�l reaction(carried out at 37 °C) consisted of 13mMHEPES, pH 7.4, buffercontaining 10mMMnCl2, 13 mMMgCl2, 50 mMKCl, 13 mg/mlbovine serum albumin, 0.7 mM phosphoenolpyruvate, 0.6 mM

NADH, 1 unit of pyruvate kinase, 1.23 units of lactate dehydro-genase, 10 mM 3-phosphoglycerate (the acceptor molecule),and 1 mM NDP-sugar. The amount of protein varied between0.5 and 2.5 �g/ml. The decrease in NADH was continuouslymeasured at 340 nm using a Fluostar Optima plate reader(BMG Labtech). Specific activity was calculated by the changeof absorbance over time using an excitation coefficient ofNADH of 6.22 mM cm�1.

Crystal Structure of a Mycobacterial Glycosyltransferase of the GT-A Fold

27882 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 41 • OCTOBER 10, 2008

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RESULTS

Crystal Structure of MAP2569c—Although Rv1208 wasinsoluble, the expression of its close homologue, MAP2569c,yielded soluble protein that was amenable for structuralstudies (10). We subsequently determined the crystal struc-ture of apo-MAP2569c using the MAD technique (see“Experimental Procedures” and Table 1). The structure wasrefined to 1.85 Å to Rfactor and Rfree values of 18.4 and 19.8%,respectively (see Table 2).The MAP2569c (329 amino acids, 34.8 kDa) protomer is

globular (�65 � 45 � 40 Å) and rich in �-helices (Fig. 1, A andB). At the core of MAP2569c is a mixed nine-stranded twisted�-sheet (sA) with topology �5-�4-�3-�6-�8-�7-�9-�2-�1 (�1and �7 are antiparallel), flanked by three helical segments oneither side. As expected from its low sequence similarity to theGT2 family, MAP2569c contained the GT-A scaffold observedin SpsA (8) (see Fig. 1, A and B) and can be subdivided into anN-terminal domain, “catalytic” domain, and C-terminaldomain.The catalytic domain of MAP2569c (residues 50–260)

exhibited the characteristic �/�/� architecture of the GT-Afold, which is subdivided into “nucleotide-binding” (residues50–138) and “acceptor-binding” (residues 142–260) subdo-mains. The nucleotide-binding subdomain ofMAP2569c com-prised the “Rossmann-like” fold (5) with four parallel �-strands(�5-�4-�3-�6), interspersed with helices (see Fig. 1, A and B).The acceptor-binding subdomain contained three antiparallel�-strands (�8-�7-�9), also separated by helices, and a disor-dered region (residues 174–188). The signature catalytic DXDmotif of the GT-A fold is found, as expected, at the juncture ofthe two catalytic subdomains and comprised Asp-139–Ser-140–Asp-141. A second small antiparallel �-sheet (sB), per-pendicular to the central �-sheet (sA), is formed by two short�-stands: �6� at the N terminus of the acceptor-binding subdo-main and�9�within a short loop bridging the acceptor-bindingsubdomain and C-terminal domain. This loop (residues 261–267) sat adjacent to the DXD motif and traced the nucleotide-binding/acceptor-binding subdomain divide. The C-terminaldomain (residues 268–329) contained a long helical region anda prominent antiparallel two-stranded twisted �-sheet (sC;�11-�10) rich in aromatic residues.Although there was a monomer in the asymmetric unit, a

crystallographic dimer was observed in which this unusualC-terminal tail wrapped around its 2-fold related partner (seeFig. 1C). The remainder of this dimeric interface is formed byresidues in the long helical region at the start of the C-terminaldomain and in the N-terminal domain and acceptor-bindingsubdomain. The buried surface area at this dimeric interfacewas quite extensive (�1900 Å2) and is therefore likely to repre-sent a biological dimer. Supporting this assertion are AUCstudies of MAP2569c, which was shown to be a dimer in aque-ous solution (see supplemental Fig. 1 and supplemental Table1). Given the location of the substrate-binding sites, it appearsthat the MAP2569c dimer is probably required for stability asopposed to catalysis. Accordingly, the dimeric MAP2569cexhibits a GT-A fold and DXD motif consistent with GTactivity. T

AB

LE1

Dat

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ion

and

ph

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tics

APS

,AdvancedPh

oton

Source

(Chicago,IL);N

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Peak

Infle

ction

Remote

MAP2

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ate

(pH

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MAP2

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(pH

7.0)

MAP2

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�Mn2

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(pH

7.0)

MAP2

569c

�UDP�Glc

(pH

5.5)

MAP2

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�UDP�GlcNAc

(pH

7.0)

Dataset

Temperature

(K)

100

100

100

100

100

100

100

100

X-ray

source

APS

,IMCA,

17ID

-DAPS

,IMCA,

17ID

-DAPS

,IMCA,

17ID

-DAPS

,IMCA,

17ID

-DAPS

,GMCA-C

AT,

23ID

-DRigaku

RU-H

3RHB

Rigaku

RU-H

3RHB

APS

,GMCA-C

AT,

23ID

-DDetector

ADSC

Mar

CCD16

5ADSC

Mar

CCD16

5ADSC

Mar

CCD16

5ADSC

Mar

CCD16

5Mar

CCD30

0R-AxisIV

��

R-AxisIV

��

Mar

CCD30

0

Wavelen

gth(Å

)0.97

930.97

940.96

411.00

001.00

531.54

181.54

181.00

53So

aktim

e(h)

NA

NA

NA

NA

66

66

Soak

conc

entration(m

M)

NA

NA

NA

NA

100

100

100

100

Spacegrou

pP4

1212

P412

12P4

1212

P412

12P4

1212

P412

12P4

1212

P412

12Celld

imen

sions

(a,c)(Å)

86.3,106

.286

.3,106

.286

.3,106

.286

.6,104

.386

.9,104

.287

.0,103

.786

.8,103

.887

.0,103

.9Re

solutio

n(Å

)2.3

2.3

2.3

1.85

2.3

3.1

2.6

2.2

No.of

observations

103,15

310

3,12

910

3,09

115

7,82

317

4,87

239

,113

56,017

199,95

4No.of

unique

observations

18,330

18,340

18,352

34,235

18,027

7555

12,684

20,893

Multip

licity

5.7

5.6

5.6

4.6

9.7

5.2

4.4

9.6

Datacompleten

ess(%)

99.6(100

)a99

.6(100

)99

.7(100

)99

.2(100

)98

.8(99.6)

99.0(99.9)

99.5(100

)99

.9(100

)�I/

�(I)

�9.7(2.9)

9.1(2.6)

10.0(2.1)

12.6(2.2)

6.2(2.2)

15.2(2.5)

11.0(2.2)

5.1(2.7)

R sym

b(%

)5.0(26.0)

5.4(29.8)

5.0(37.2)

3.3(35.3)

6.6(34.7)

3.6(30.6)

4.6(34.8)

7.3(28.6)

MADan

alysis

Selenium

sites

3FO

MMADc

0.58

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Comparison of the GT-A Fold of MAP2569c with OtherGlycosyltransferases—We next compared the GT-A fold ofMAP2569c to other structures in the PDB using the DALIserver (21) and identified a number of nucleotide-binding pro-teins with similar architecture. The closest structural match(with a Z-score of 18.6) was to the catalytic domain of manno-sylglycerate synthase (MGS) from Rhodothermus marinus (22)(ProteinData Bank code 2bo4, 181C� atoms; rootmean squaredeviation of 2.0 Å, and 22% sequence identity). Based onsequence similarity,MGSwas previously classified as amemberof the GT2 family (23); however,MGSwas subsequently shownto use a retainingmechanism andwas reclassified as the found-ing member of the GT78 family (22). The next two closeststructural homologues from theGT superfamily (withZ-scoresof 13.5 and 12.4, respectively) belong to SpsA fromB. subtilis (8)(ProteinData Bank code 1qg8, 166C� atoms, rootmean squaredeviation of 3.4 Å and 13% sequence identity) and polypeptideN-acetylgalactosaminyltransferases from Homo sapiens (24)(Protein Data Bank code 2ffu, 171 C� atoms, root mean squaredeviation of 3.0 Å, and 16% sequence identity). As with MGS,polypeptide N-acetylgalactosaminyltransferase was initiallygrouped with SpsA in the GT2 family but has since been shownto use a retainingmechanism andwas subsequently reclassifiedas a member of the GT27 family (19, 21). Of the 20 higheststructural matches to the GT-A fold of MAP2569c, only six areGTs. Consequently, theGT-A fold ofMAP2569c shows higheststructural homology to the catalytic domain of a GT with aretainingmechanism and displays significant structural homol-ogy to other nucleotide-binding protein families. This struc-tural similarity of MAP2569c to other nucleotide-binding pro-tein families is indicative of both the structural diversity of andlack of structural data for this rapidly growing class of enzymes.Nucleotide Binding—As a candidate member of the GT2

family, MAP2569c is predicted to use Mn2� and a nucleotide-sugar as its activated donor substrate (23). Accordingly, wesought to define the nucleotide specificity of this enzyme. First,we screened for nucleotide binding via a crystallographicapproach. A crystallography-based approach has previously been

applied with other GTs to identify the donor sugar (25). Namely,we soaked 10mMCMP, GDP, and UDP nucleotides into the apo-crystal form. The difference Fourier electron density maps calcu-lated using these data (statistics not shown) revealed evidence forUDP only and not for CMP or GDP at the predicted donor sub-strate site of MAP2569c and thus indicated that UDP is the likelynucleotide component of the donor substrate forMAP2569c.We subsequently determined the 2.3 Å resolution structure

of MAP2569c in complex with its donor substrate product,Mn2��UDP, at pH 7.0 and refined it to Rfactor and Rfree values of19.3 and 21.7%, respectively (see “Experimental Procedures”)(Tables 1 and 2 and Fig. 2A). Overall, the structure ofMAP2569cis unchanged on binding UDP, although a major conformationalchange was observed in the loop (residues 261–267) linking theacceptor-binding subdomain andC-terminal domain (see below).The�- and�-phosphates onUDP interactwithAsp-141O�2 of theDXD motif via the Mn2� ion (Fig. 3A), as observed previously inthe SpsA structure (6). In addition,His-263N�1 on the loop linkingthe acceptor-binding subdomain and C-terminal domain alsocoordinates theMn2� ion.The�-phosphate alsohydrogen-bondsto Tyr-234O�, and the �-phosphate forms a water-mediatedhydrogen bond with Asp-139O�2 and is further within van derWaals contact of the side chains ofMet-274 and Arg-266.The uracil base of UDP stacked against the side chains of

Leu-57 and of Lys-119 on the �-helix hF and made further vander Waals contacts with the side chains of Pro-55 and Ser-86.The O2 on the uracil hydrogen-bonds to one of the two alter-nate conformations modeled for Ser-86O�. In addition to thesecontacts with the nucleotide-binding subdomain, the uracil isalso in van derWaals contact with Tyr-234 on the 310-helix hL.The ribose ring of UDP formed van der Waals contacts with

the side chains of Pro-55, Lys-119, andTyr-234. TheO2* on theribose hydrogen-bonds to Leu-57N and the carboxylates ofGlu-59, and the O3* hydrogen-bonds to Pro-55O. The ribose ringfurther interacts with the conserved DXD motif, with the O3*also hydrogen-bonding to the Ser-140N,O�. Thus, the largenumber of contacts between MAP2569c and UDP provided abasis for its specificity for this nucleotide.

TABLE 2Refinement statistics

Model MAP2569c�Citrate(pH 5.5)

MAP2569c�Mn2��UDP(pH 7.0)

MAP2569c�Mn2��UDP-Glc(pH 7.0)

MAP2569c�UDP-Glc(pH 5.5)

MAP2569c�UDP-GlcNAc(pH 7.0)

Nonhydrogen atomsProtein 2289 2255 2252 2289 2294Ligand/ion 42 31 42 38 38Water 102 36 4 24 48Resolution range (Å) 30–1.85 30–2.3 30–3.1 30–2.6 30–2.2Rfactor/Rfree

a (%) 18.4/19.8 19.3/21.7 19.6/23.4 18.0/21.9 18.8/20.5Root mean square deviation

from ideal valuesBond lengths (Å) 0.015 0.016 0.016 0.015 0.015Bond angles (degrees) 1.452 1.505 1.298 1.622 1.414

Ramachandran plot (%)Most favored regions 91.5 91.5 89.8 90.2 91.9Additional allowed regions 8.5 8.5 9.4 9.4 8.1

B-factors (Å2)Average main chain 27.0 45.5 42.0 29.8 44.7Average side chain 28.9 46.3 42.4 31.0 45.6Average ligand/ion 61.4 61.4 88.1 42.5 65.2Average water molecule 42.5 46.4 53.2 42.4 42.8

aRfactor � hkl�FP(obs)� � �FP(cal)�/hkl�FP(obs)� for all reflections. For refinement of the apo crystal form, Rfree was calculated using randomly selected reflections (10%). Forrefinement of the ligated MAP2569c complexes, the same Rfree data set was used as selected in the apo crystal form.



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MAP2569c Complexed with UDP-glucose—To identify whatUDP-sugar MAP2569c could bind, a similar co-crystallizationapproach was employed. UDP-activated sugars that act asnatural donor substrates for GTs include UDP-GalNAc, UDP-GlcNAc, UDP-�-L-arabinose, UDP-Gal, UDP-Glc, UDP-�-D-glucuronic acid, and UDP-�-D-xylose (although only UDP-GlcNAc, UDP-Gal, and UDP-Glc have been found inmycobacteria, all natural UDP-sugar donor substrates wereinvestigated for comparison). For all crystal structures solved

using these UDP-sugars, there waselectron density for at least the ura-cil ring of the UDP-sugar at the pre-dicted donor substrate site ofMAP2569c. For example, the com-plex with UDP-GlcNAc was refinedto 2.2 Å to Rfactor and Rfree values of18.8 and 20.5%, respectively (see“Experimental Procedures” andTables 1 and 2) and revealed evi-dence for the uracil and ribose ringsand �-phosphate of the UDP-sugar(see supplemental Fig. 2). However,only in the complex with UDP-Glcwas electron density also observedfor Mn2�, the �-phosphate, and thesugar moiety (see below) (Fig. 2B) atthe predicted donor substrate site.This suggests specificity for UDP-Glc over all other natural, includingchemically related, UDP-sugars.Second, to further verify this

specificity, the enzyme activity ofMAP2569c was analyzed with theUDP-sugars found in mycobacteria,UDP-GlcNAc, UDP-Gal, and UDP-Glc. The highest activity wasobserved with UDP-Glc, with noactivity toward UDP-Gal or UDP-GlcNAc (Fig. 3), suggesting thatMAP2569c uses UDP-Glc as its nat-ural donor sugar. There was someresidual activity observed withGDP-Glc (20%of that observedwithUDP-Glc), suggesting that there issome plasticity in donor sugar bind-ing (Fig. 3).UDP-Glc sat in a “folded back”

conformation at the putative donorsite, reminiscent of the conforma-tion first observed in a donor sub-strate analogue in complex with theretaining GT LgtC (26), with a bur-ied surface area of �50 Å2. Thedonor substrate traverses the cleftbetween the nucleotide- andacceptor-binding subdomains (seeFig. 1B). The interactions withUDP are similar to those observed

in the complex of MAP2569c with Mn2��UDP.The Glc moiety of the donor substrate is within hydrogen

bonding distance of several residues within MAP2569c (seebelow) (Figs. 2C and 4). The O4� and O6� OH moieties hydro-gen-bond to Glu-237O�1 on the �-helix hP; the O3�-OH andO4�-OHhydrogen-bond to Lys-119N, and theO3�-OH furtherhydrogen-bonds to Asp-139O�2. In addition, Asp-139O�2 formsa water-mediated hydrogen bond with the O2�-OH. TheO5�-OH hydrogen-bonds to Leu-214O, and the O6�-OH fur-

FIGURE 1. Ribbon representations of the structure of MAP2569c. MAP2569c is shown in the same orienta-tion in complex with citrate (A) and Mn2��UDP-Glc (B) and colored according to domain or subdomain. Disor-dered regions are indicated by dashed lines. Secondary structure was calculated using DSSP (34). The N-termi-nal domain is colored pink, the Rossmann domain is shown in cyan, the catalytic DXD motif is shown in red, theacceptor-binding domain is shown in green, the flexible loop is shown in yellow, the long helical region is inpurple, and the remainder of the C-terminal domain (residues 286 –329) is shown in salmon. The ligands areshown in a ball and stick representation, with citrate superimposed in the complex with Mn2��UDP-Glc. C, thedimer. The 2-fold symmetry-related molecules are shown in pink and yellow. Disordered regions are indicatedby dashed lines. This figure and all figures in this paper were drawn using PyMOL (35).



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ther forms water-mediated contacts with Tyr-234O andIle-238N. Given that the O4�-OH is the only distinctionbetweenUDP-Glc andUDP-Gal, the interactionswith this oxy-gen are probably important for discriminating the naturaldonor substrate forMAP2569c. The C6� andO6�make van derWaals contacts with the side chain of Leu-214, and the C5� andO5� also make van der Waals contacts with the side chain ofMet-274, which adopts a different conformation to accommo-date the Glc moiety. Consequently, the sugar moiety of thedonor substrate forMAP2569c interacts with residues from thecatalytic and C-terminal domains of MAP2569c, and the largenumber of contacts dictates the specificity for UDP-Glc.Induced Fit—The crystal structures of GTs of the GT-A

fold have often indicated that a flexible loop in the vicinity ofthe nucleotide-binding site plays a critical role in the cata-lytic mechanism of the enzyme (for a review, see Qasba et al.(27)). To investigate whether induced fit plays a role inMAP2569c, we compared the structures of the apo and

ligated forms. The comparativeanalyses revealed that upon liga-tion, the loop (residues 261–267)linking the catalytic and C-termi-nal domains in MAP2569cchanges conformation.In the apo form of MAP2569c,

residues 262–263 on the flexibleloop form a short �-strand (�9�) ofthe “transient”�-sheet (sB,�6�-�9�)and residues 268–270 form a 310-helix (hO) at the start of the C-ter-minal domain (see Fig. 5). How-ever, in the MAP2569c�Mn2��UDP-Glc complex, this flexibleloop adopted a different conforma-tion, driven by the participation ofthe imidazole group on His-263 inaMn2�-mediated interaction withthe �- and �-phosphates on UDP-Glc (see Fig. 5). To accommodatethis, the �9� and �6� strands arerestructured into loops, and resi-dues 268–270 form an �-helix.

In the complex of MAP2569c�Mn2��UDP, the loop adopted an“intermediate” conformation (rootmean square deviation of�0.4 Å) tothat observed in the apo and “donorsubstrate” liganded forms ofMAP2569c. Moreover, the �-phos-phate on UDP interacted less withthe C-terminal domain than in thecomplex with Mn2��UDP-Glc.Instead, the �-phosphate made vander Waals contacts with the sidechain of Arg-266 (disordered in theapo and Mn2��UDP-Glc ligandedforms of MAP2569c) and is withinwater-mediated hydrogen bond-

ing distance of Asp-139O�2. Consequently, the conforma-tional changes observed in the flexible loop containing His-263 and associated changes in adjacent secondary structuralelements are important for coordination of the donor sub-strate and for stabilization of the donor substrate product ofMAP2569c.The Role of His-263—To further investigate the involvement

of His-263 in Mn2� coordination, we determined the crystalstructure ofMAP2569c in complexwithMn2� andUDP-Glc ata pH value below the pKa of histidine (pH 5.5 was used in thisstudy). At such a pH value, histidine acts as an acid rather thanas a base (as under physiological conditions) and thus cannotparticipate in Mn2� coordination. The complex withMn2��UDP-Glc at pH5.5was refined to 2.6Å toRfactor andRfreevalues of 18.0 and 21.9%, respectively (see “Experimental Pro-cedures”) (Tables 1 and 2). The structure revealed evidence forthe uracil and ribose rings and �-phosphate of the UDP-sugaronly (see Fig. 2C), indicating the requirement for the ionizable

FIGURE 2. Unbiased electron density for the putative donor substrate product and donor substrate ofMAP2569c. A, Mn2��UDP; B, Mn2��UDP-Glc at pH 7.0; C, UDP-Glc at pH 5.5. A–C, the electron density mapsshown are Fo � Fc simulated annealing omit maps contoured at 3�. The Mn2� ion and water molecules arerepresented by purple and red spheres, respectively. Hydrogen bonds and Mn2�-mediated interactions arerepresented by dashed black and purple lines, respectively.



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imidazole group on His-263 for Mn2� coordination, and thusalso for �-phosphate and sugar coordination, in the complex ofMAP2569c with Mn2��UDP-Glc at pH 7.0.Comparison with Other GTs—Of the inverting GTs of the

GT-A fold solved in complex with a nucleotide-sugar donorsubstrate, the catalytic domain of MAP2569c showed the high-est structural homology to N-acetylglucosaminyltransferase Ifrom Oryctolagus cuniculus (28) (Z-score 12.0, Protein DataBank code 1foa, 164 C� atoms superimpose with a root meansquare deviation of 3.2 Å and a sequence identity of 13%) (21).Although the flexible loop inN-acetylglucosaminyltransferase Ialso undergoes conformational change on activated donor sub-strate coordination, no residue on this loop is directly involvedin divalent metal coordination. In addition, rather than trace

the nucleotide-binding/acceptor-binding subdomain divide,this loop in N-acetylglucosaminyltransferase I forms a “flap”over its nucleotide-sugar substrate (see Fig. 6A).Next we compared the residues involved in donor substrate

coordination in MAP2569c with those found in other GTs ofthe GT-A fold and identified structurally homologous seg-ments in the “retaining” GTs MGS (22) and polypeptideN-acetylgalactosaminyltransferase (24). In all three structures,a histidine (as part of an Asp/His motif) within this loop isrequired for divalent metal ion coordination. In addition, thereis a similar long helical region at the start of the C-terminaldomain. In MAP2569c and MGS, structurally homologousmethionine side chains in this region (Met-274 in MAP2569candMet-229 inMGS) have also been shown (here) to play anal-ogous roles in sugar coordination (see Fig. 6B).The folded back conformation of UDP-Glc in complex with

MAP2569c also resembled the conformation of GDP-Man incomplex with MGS, and the mode of sugar coordination inthese structures is similar (see Fig. 4) The side chains of Leu-163, Trp-189, and Met-229 in MGS overlay with those of Leu-214, Tyr-234, and Met-274 in MAP2569c and similarly formhydrophobic contactswith the sugarmoiety in these structures.The side chains of Asp-192 inMGS and Glu-237 inMAP2569care both within hydrogen bonding distance of the O4� and O6�

FIGURE 3. NDP-sugar specificity for MAP2569c. Relative activity ofMAP2569c in the presence of different donor NDP-sugars and the acceptormolecule, 3-phosphoglycerate (33). The results are expressed as relativeactivity of the highest value and are the mean S.D. of three experimentsperformed in triplicate.

FIGURE 4. Comparison of the donor substrate binding site of MAP2569cto that found in MGS. MGS (pink) in complex with Mn2��GDP-Man (donorsubstrate not shown) superposed on MAP2569c (green) in complex withMn2��UDP-Glc. The hydrogen-bonding network spanning Glu-237 to Asp-139 is represented by dashed black lines. Other hydrogen bonds and Mn2�-mediated interactions observed in the MAP2569c�Mn2��UDP-Glc complex atpH 7.0 are represented by dashed gray and purple lines, respectively.

FIGURE 5. Comparison of the flexible loop of MAP2569c in complex withcitrate, Mn2��UDP, and Mn2��UDP-Glc at pH 7.0. The shift in the flexibleloop containing His-263 (shown in a ball and stick representation) and associ-ated conformational changes in adjacent secondary structural elementsbetween MAP2569c in complex with citrate (in salmon), Mn2��UDP (in cyan),and Mn2��UDP-Glc at pH 7.0 (in green) reshape the “active site” of MAP2569c.



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on the sugar. In addition, Glu-237 inMAP2569c is involved in ahydrogen bonding network via Lys-119 that extends to the firstaspartate of the DXDmotif. This network, also observed in thestructures of the retainingGTs LgtC (26) andMGS (22) in com-plex with Mn2��UDP-2-deoxy-2-fluoro-Gal (a donor substrateanalogue) andMn2��GDP-Man, respectively, involves the basicand acidic side chains of Lys-119 (Lys-76 inMGS) and Asp-139(Asp-100 inMGS), both of which are within hydrogen bondingdistance of theO3� (see Figs. 3B and 4). Themain chain carbon-yls on Leu-163 in MGS and Leu-214 also participate in analo-gous hydrogen-bonding interactions with the sugar moiety.

However, additional residues onthe flexible loop in MGS, which isnotably partially disordered in thecomplex of MGS with Co2��GDP,participate in Mn2��GDP-Mancoordination, with Arg-218 andTyr-220 also interacting with the�- and �-phosphates on GDP-Man, respectively (see Fig. 6B).The structural homology observedbetween MAP2569c and retainingGTs of the GT-A fold extendsbeyond the catalytic domains ofthese enzymes to the flexible loopsand adjacent secondary structuralelements that undergo significantconformational change on donorsubstrate coordination.The Acceptor Binding Site of

MAP2569c—A bound organic mol-ecule often indicates an important(functional) site in a crystal struc-ture. Indeed, the binding site of acitrate molecule in the apo form ofMAP2569c is similarly located tothe binding site observed for a cit-rate in the apo formofMGS (see Fig.7, A and B), with Thr-192 and Arg-261 in MAP2569c and Thr-139 andArg-131 in MGS playing analogoushydrogen-bonding roles in coordi-nating the citrate in these struc-tures. Indeed, the citrate in MGSnotably makes interactions withMGS similar to that of the naturalacceptor substrate of MGS, D-glyc-erate, indicating the adaptability ofthis binding site. Although thethreonines within hydrogen bond-ing distance of the citrate are fromstructurally homologous segmentsof MAP2569c and MGS, the argi-nine residues are from differentregions. To illustrate, Arg-261 inMAP2569c is from the flexible loopinvolved in “substrate donor” coor-dination, whereas Arg-131 in MGS

is from a loop that structurally corresponds to the disorderedregion (residues 174–188) in MAP2569c. Consequently,although the citrate binding sites in the apo form ofMAP2569cand MGS are similarly located, the arginines within hydrogenbonding of the citrate in MAP2569c and MGS are from non-structurally homologous segments of the polypeptide chain,suggesting that the mode of natural acceptor substrate bindingmay differ between MAP2569c and MGS.Sequence Similarity of MAP2569c to Putative Orthologues

from the Corynebacterineae—To gain further insight into thefunction of MAP2569c, we compared the sequence of

FIGURE 6. Comparison of the flexible loop of MAP2569c in complex with its putative donor substrate andthose of inverting and retaining GTs of the GT-A fold. A, N-acetylglucosaminyltransferase I (in purple) incomplex with Mn2��UDP-GlcNAc (donor substrate not shown) superposed on MAP2569c (in green) in complexwith Mn2��UDP-Glc (shown in a ball and stick representation). B, MGS (in pink) in complex with Mn2��GDP-Man(donor substrate not shown) superposed on MAP2569c (green) in complex with Mn2��UDP-Glc (shown in a balland stick representation). The Mn2�-mediated interactions between the N�1 on His-263 of MAP2569c and the�- and �-phosphates on UDP-Glc are represented by dashed purple lines.

FIGURE 7. Comparison of the citrate binding site of MAP2569c to that found in MGS. A, MAP2569c; B, MGS.For both structures, the Rossmann subdomain is shown in cyan, the catalytic DXD motif is shown in red, theacceptor-binding subdomain is shown in green, the flexible loop is shown in yellow, the structurally homolo-gous helical region at the start of the C-terminal domain is shown in purple, and the remainder of the C-terminaldomain is shown in salmon. The residues with side chains involved in direct hydrogen-bonding interactionswith the bound solvent molecule are also shown in ball and stick representations. The electron density shownfor citrate is a final 2Fo � Fc synthesis contoured at 1�. In MGS, Thr-139 and Arg-131 also form direct hydrogenbond interactions with the natural acceptor substrate D-glycerate.



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MAP2569c with those of its putative orthologues from closelyrelated Mycobacterium species, including Mycobacteriumulcerans,M. tuberculosis, andMycobacterium leprae, and fromother representatives of the Corynebacterineae, includingNocardia farcinica andCorynebacterium glutamicum (see sup-plemental Fig. 3). The sequence identity to MAP2569c, calcu-lated using ClustalW (29), of these orthologues ranges from 47to 85%,with a high level of identity for all sequences throughoutthe catalytic domain (residues 50–260), flexible loop (residues261–267), and long helical region at the start of the C-terminaldomain (residues 268–285) of MAP2569c, suggesting thatthese orthologues from the Corynebacterineae share the samearchitecture for these structural modules involved in donorsubstrate coordination in MAP2569c.In addition, apart from Ser-86 and Ser-140, all of the

MAP2569c residues observed to form (direct or indirect)hydrogen-bonding, Mn2�-mediated, or van derWaals interac-tions with UDP-Glc at pH 7.0 are conserved in these ortho-logues. Moreover, the citrate-binding site in MAP2569c is wellconserved throughout the orthologues. This essentially invari-ant nature of the donor and potential acceptor substrate bind-ing sites inMAP2569c across the orthologues from theCoryne-bacterineae suggests that each of these candidateGTs performsa similar role in the synthesis of the unique cell wall of thissuborder.

DISCUSSION

The ubiquitous nature of oligosaccharides and glycoconju-gates and the rapidly growing number of candidate GTsequences uncovered by genomic analyses are indicative of awealth of untapped knowledge on carbohydrate function. Dueto the distant sequence similarities between some GT families,crystal structure analysis, as exemplified here, presents a pow-erful tool for establishing the relatedness between GTs andhelps to bridge the chasm between the vast amount of sequencedata and the paucity of functional and mechanistic knowledgeabout this important class of enzymes.We have determined the structure ofMAP2569c, which pos-

sessed a GT-A fold and a DXDmotif associated with GT2 fam-ily activity. MAP2569c contains a flexible loop, which under-goes conformational change on activated donor substratebinding and houses a histidine residue required for divalentmetal ion coordination, as has also been observed in the invert-ingGT,�4-galactosyltransferase (30). However, the overall foldand catalytic core ofMAP2569c exhibits highest structural sim-ilarity to MGS, a GT that has been shown to use a retainingmechanism and represents the archetype of the GT78 family(22). Moreover, the structural homologies observed betweenMAP2569c and retaining GTs of the GT-A fold extend beyondthe flexible loop in the vicinity of the activated donor substratebinding site to the adjacent secondary structural elements thatalso undergo conformational change on activated donor sub-strate coordination. Through crystallographic and enzymaticanalyses, we revealed MAP2569c preferentially binds the acti-vated donor substrate Mn2��UDP-Glc. The interactionsbetween MAP2569c and Mn2��UDP-Glc are also reminiscentof those observed between MGS and its activated donor sub-

strate (22). This level of specificity of MAP2569c is ratherunusual in comparison with the GTs in general.GTs use a carboxylate residue to bind the acceptor substrate

and initiate transfer of the sugarmoiety (27). The acidic residuein the retaining GTMGS (Asp-192) is structurally conserved inMAP2569c (Glu-237).Moreover, these residues are involved insimilar hydrogen-bonding interactions at the donor substratesites of MAP2569c and MGS. Nevertheless, as noted by Flintet al. (22), the acidic residue is similarly located in GTs of theGT-A fold that use both inverting and retaining mechanisms,possibly reflecting the evolution of retaining GTs of the GT-Afold from the inverting GT2 family (31). Flint et al. (22) alsosuggested that a change in the angle of the corresponding�-helices containing the acidic group in inverting versus retain-ing GTs of the GT-A fold could define the mechanism of theseenzymes. However, when comparing MAP2569c and otherGTs, we could find no such correlation (data not shown).Rather, one shared feature observed in retaining GTs of theGT-A fold is the position of the side chain of the acidic residueon the nucleoside-proximal side of the sugar moiety, asopposed to the nucleoside distal side of the sugar moiety, asobserved in inverting GTs of the GT-A fold (see supplementalFig. 4) (32). Although there are no clear structural features thatdefine themechanismofGTs, based on the accumulative struc-tural homologies between MAP2569c and MGS, we suggestthat MAP2569c possesses the same retaining mechanism ofMGS, but this contention will require further experimentation.We also provide evidence that the orthologues ofMAP2569c

from other Mycobacterium sp. and from related Corynebacte-rineae share a common architecture. Moreover, given that theresidues involved in donor and acceptor substrate recognitionare also conserved across the orthologues, we suggest that thesealso exhibit similar substrate specificities and hence perform asimilar role in the biosynthesis of the unique cell wall of thesebacteria. Although all known and characterized GTs of theGT-A fold found inMycobacterium species aremembers of theinverting GT2 family (3), our analyses indicate that some GTsmay need to be reclassified.A recent report describes glycosyl-3-phosphoglycerate syn-

thase from Mycobacterium smegmatis and Mycobacteriumbovis that has homology (�25% sequence identity) to the M.tuberculosis H37Rv gene Rv1208. Glucosyl-(1–2)-glycerate isfound at the reducing end ofmethylglucose lipopolysaccharide,which is involved in regulating fatty acid synthesis inMycobac-terium (33). The formation of glucosylglycerate is performed byglycosyl-3-phosphoglycerate synthase, inwhich it transfers glu-cose fromNDP-glucose to glucosyl-3phophoglycerate. Recom-binant glycosyl-3-phosphoglycerate synthase from M. bovisshowed optimal activity with UDP-Glc and strictly requiredMg2�. Glycosyl-3-phosphoglycerate synthase enzymes areclassified into the retaining family 81 of GTs. These observa-tions lend support to MAP2569c (and Rv1208) being a UDP-Glc specific GT that uses a retaining mechanism.Given the unique complex carbohydrate content of the

Mycobacterium cell wall, GTs involved in its biosynthesis pres-ent promising targets for new drugs in the treatment of tuber-culosis and other diseases caused by Mycobacterium species.Previous studies using saturation mutagenesis screening have



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suggested that theM. tuberculosis homolog Rv1208 is essentialfor survival and growth (9). The function of Rv1208 is not yetknown, but its most likely role is as a putative GT inMycobac-terium cell wall biosynthesis. Its high specificity for its candi-date activate donor substrate together with a natural acceptorsubstrate that is probably exclusively found in Corynebacteri-neae makes the orthologues of MAP2569c promising targetsfor new antimicrobials.

Acknowledgments—We thank the IMCA and GM/CA-CAT staff forassistance in data collection at the Advanced Photon Source (Chicago,IL).

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