Crystal Structure of a UDP-glucose-specific Glycosyltransferase from a Mycobacterium Species

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.

Crystal Structure of a UDP-glucose-specific Glycosyltransferase 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储, 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 Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, the ¶Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, the 储Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, and the **Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a devastating bacterial pathogen, and infection by M. tuberculosis continues to be common particularly in developing countries, with particular strains becoming resistant to multiple frontline drugs. The pathogenicity of M. tuberculosis is partly attributable to its waxy cell wall that

* This work was supported by the Australian Research Council (ARC) Centre of Excellence in Structural and Functional Microbial Genomics and the National Health and Medical Research Council (NHMRC) of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” 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) contains supplemental 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 Collaboratory 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: jamie.rossjohn@med. monash.edu.au. 2 A NHMRC Career Development Award Fellow. To whom correspondence may be addressed. Tel.: 613-9905-3736; Fax: 613-9905-4699; E-mail: [email protected].

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consists of covalently linked layers of peptidoglycan, arabinogalactan, and mycolic acids (for a review, see Crick et al. (1)), interspersed with lipids and glycolipids, and capped with polysaccharides (2). Despite the major importance to the biology of Mycobacterium sp. and other Corynebacterineae, the biosynthetic pathways for many of these carbohydrates are poorly understood, although it is established that the most prolific class of enzymes involved in these pathways are the glycosyltransferases (GTs)3 (3). Indeed, there are 41 known or putative GTs in M. tuberculosis strain H37Rv listed in the CAZy (carbohydrate-active enzyme) data base, representing over 1% of the 3900 open reading frames in the M. tuberculosis H37Rv genome (4). Ethambutol, a front line anti-tuberculosis drug, targets the EmbA and EmbB GTs that synthesize the arabinogalactan cell wall layer, thereby marking GTs as promising new drug targets. GTs transfer sugar moieties from the activated donor substrate, mostly in the form of a nucleoside-diphospho-sugar, to a myriad of specific acceptor substrates to generate innumerable oligosaccharide and glycoconjugate products that are often required for species- or cell specific processes. GTs are classified as either “inverting” or “retaining,” depending on whether the stereochemistry of the anomeric carbon is retained or inverted in the product relative to that in the donor substrate. Moreover, despite the sequence diversity within the GT superfamily, GT structures solved to date adopt one of two common folds, termed “GT-A” or “GT-B.” Both of these folds adopt the “Rossmann-like fold,” with the GT-A fold containing a single Rossman-like or nucleotide-binding domain (5) and the GT-B fold containing two similar Rossmann-like domains. Moreover, members within a particular GT family are predicted to share the same inverting or retaining mechanism and GT-A or GT-B fold. Furthermore, GTs of the GT-A fold that utilize a nucleoside-diphospho-sugar typically contain a “DXD” (or XDD or EXD) motif that is involved in metal ion-mediated activated donor substrate coordination and is also required for catalytic activity (6). In contrast to the abundance of GT sequences available, there is a lack of functional, mechanistic, and structural data on 3

The abbreviations used are: GT, glycosyltransferase; MGS, mannosylglycerate synthase; MAD, multiple anomalous dispersion.

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Glycosyltransferases (GTs) are a large and ubiquitous family of enzymes that specifically transfer sugar moieties to a range of substrates. Mycobacterium tuberculosis contains a large number of GTs, many of which are implicated in cell wall synthesis, yet the majority of these GTs remain poorly characterized. Here, we report the high resolution crystal structures of an essential GT (MAP2569c) from Mycobacterium avium subsp. paratuberculosis (a close homologue of Rv1208 from M. tuberculosis) in its apo- and ligand-bound forms. The structure adopted the GT-A fold and possessed the characteristic DXD motif that coordinated an Mn2ⴙ ion. Atypical of most GTs characterized to date, MAP2569c exhibited specificity toward the donor substrate, UDP-glucose. The structure of this ligated complex revealed an induced fit binding mechanism and provided a basis for this unique specificity. Collectively, the structural features suggested that MAP2569c may adopt a “retaining” enzymatic mechanism, which has implications for the classification of other GTs in this large superfamily.

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

EXPERIMENTAL PROCEDURES Production and Crystallization of MAP2569c—Detailed methods used for the cloning, overexpression, purification, and crystallization of MAP2569c have been published previously (10). Briefly, selenomethionine-labeled protein was overexpressed in Escherichia coli B834 (DE3) grown in autoinducing medium, as described by Studier (11) at 25 °C for 24 h. The crystallized form of MAP2569c in this study comprised residues 5–329 of the native sequence. MAP2569c crystallized in space group P41212 with unit cell dimensions a ⫽ b ⫽ 86.6 Å and c ⫽ 104.3 Å. There is one monomer in the asymmetric unit corresponding to a solvent content of ⬃54% (v/v). The co-complex crystals were obtained by soaking the “native” crystals in 1 M ammonium sulfate, 0.1 M HEPES, pH 7.0, or 0.1 M sodium citrate, pH 5.5, 20 mM manganese chloride, 1 mM sodium thiosulfate, 5% (v/v) glycerol, and 100 mM UDP or UDP-sugar for 6 h. The crystals were prepared for x-ray data collection by the addition of 15% (v/v) glycerol prior to flash cooling directly in liquid nitrogen. X-ray Diffraction Data Collection—X-ray diffraction data were collected from crystals at 100 K on IMCA beamline 1710-D at the Advanced Photon Source, Argonne National Laboratory (Chicago, IL), using a MAR CCD 165 detector, at General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) beamline 23 ID-D at the Advanced Photon Source using a MAR CCD 300 detector, and in house using a Rigaku RU-H3RHB rotating anode generator and R-Axis IV⫹⫹ detector (see Table 1). X-ray

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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 to obtain the initial phases from selenomethionine-substituted crystals of apo-MAP2569c. Three selenium atom sites were located, and initial phases were calculated using BnP (13). The figure of merit was 0.585 to 2.3 Å (see Table 1). The initial phases were extended to 1.8 Å resolution with automated density modification using DM (14). The starting model was built into the density-modified electron density map using ARP/ wARP (15). The final model was obtained after iterative cycles of manual model building with COOT (16) and TLS and restrained refinement using REFMAC (14, 17). The final model consists of two contiguous polypeptide chains and comprised 300 residues (residues 15–173 and 189 –329 of the native sequence) and 102 water molecules. The structure was refined to 1.85 Å to Rfactor and Rfree values of 18.4 and 19.8%, respectively (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 maps revealed the location of the nucleotide or nucleotide-sugar molecules, and the subsequent structures were refined using similar protocols to those listed above (see Tables 1 and 2). For refinement of the ligated MAP2569c complexes, the same Rfree data set was used as selected in the apo crystal form. Analytical Ultracentrifugation Analysis—Sedimentation velocity experiments were conducted in a Beckman model XL-I analytical ultracentrifuge at a temperature of 20 °C. A sample of MAP2569c (380 ␮l, 1.5 mg/ml) solubilized in 20 mM Tris, 150 mM NaCl, pH 8.0, and reference (400-␮l) solutions were loaded into a conventional double sector quartz cell and mounted in a Beckman four-hole An-60 Ti rotor. Data were collected in continuous mode at 295 nm using a rotor speed of 40,000 rpm, a time interval of 300 s, and a step size of 0.003 cm without averaging. Solvent density (1.005 g/ml at 20 °C) and viscosity (1.021 cp) as well as estimates of the partial specific volume of MAP2569c (0.74 ml/g) were computed using the program SEDNTERP (18). Sedimentation velocity data at multiple time points were fitted to a single discrete species or a continuous size distribution model (19) using the program SEDFIT, which is available on the World Wide Web. Enzyme Activity—The nucleotide-sugar specificity of MAP2569c was determined by a continuous linked enzyme assay described by Grosselin et al. (20). The 200-␮l reaction (carried out at 37 °C) consisted of 13 mM HEPES, pH 7.4, buffer containing 10 mM MnCl2, 13 mM MgCl2, 50 mM KCl, 13 mg/ml bovine serum albumin, 0.7 mM phosphoenolpyruvate, 0.6 mM NADH, 1 unit of pyruvate kinase, 1.23 units of lactate dehydrogenase, 10 mM 3-phosphoglycerate (the acceptor molecule), and 1 mM NDP-sugar. The amount of protein varied between 0.5 and 2.5 ␮g/ml. The decrease in NADH was continuously measured at 340 nm using a Fluostar Optima plate reader (BMG Labtech). Specific activity was calculated by the change of absorbance over time using an excitation coefficient of NADH of 6.22 mM cm⫺1. VOLUME 283 • NUMBER 41 • OCTOBER 10, 2008

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these enzymes. This paucity of biochemical data reflects the difficulty in characterizing enzymes with countless possible combinations of donor and acceptor substrates and products. Indeed, only 29 of the 90 GT families have representative structures solved. The vast majority of sequences uncovered by genomic analyses that show distant sequence identity to known GTs are thus grouped into large “polyspecific” families. For example, the rapidly growing GT2 family is the largest such family, with over 10,000 known and candidate members, and is considered the ancestral inverting family from which all GTs that share the GT-A fold have evolved (7). The spore coat polysaccharide biosynthesis protein (SpsA) from Bacillus subtilis (8), which adopts the GT-A fold, remains the only representative of the GT2 family whose structure is solved. Due to the versatility of the GT-A fold, “rules” for discerning the substrate specificity and discriminating the mechanism of GTs that exhibit this fold remain elusive. To further our understanding of this family of GTs and to characterize new anti-tuberculosis drug targets, we investigated Rv1208, an essential enzyme (9) implicated in the biosynthesis of the oligosaccharide- and glycoconjugate-rich M. tuberculosis cell wall. As a candidate member of the GT2 family, Rv1208 is predicted to use an inverting mechanism, Mn2⫹ and a nucleoside-diphospho-sugar as its activated donor substrate. We have determined the crystal structure of MAP2569c from Mycobacterium avium subsp. paratuberculosis, a close homologue (83% sequence identity) to Rv1208. We reveal that MAP2569c possesses the GT-A fold and exhibits specificity toward the donor substrate, UDP-glucose.

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

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1.0053 6 100 P41212 87.0, 103.9 2.2 199,954 20,893 9.6 99.9 (100) 5.1 (2.7) 7.3 (28.6)

c

a

b

3 0.585 MAD analysis Selenium sites FOMMADc

Wavelength (Å) Soak time (h) Soak concentration (mM) Space group Cell dimensions (a, c) (Å) Resolution (Å) No. of observations No. of unique observations Multiplicity Data completeness (%) 具I/␴(I)典 Rsymb (%)

Detector

Statistics shown in parentheses are for the highest resolution shell. Rsym ⫽ ⌺h ⌺l兩Ihl ⫺ 具Ih典兩/⌺h⌺l 具Ih典, where Il is the lth observation of reflection h and 具Ih典 is the weighted average intensity for all observations l of reflection h. Figure of merit after BnP (13) phasing.

1.5418 6 100 P41212 86.8, 103.8 2.6 56,017 12,684 4.4 99.5 (100) 11.0 (2.2) 4.6 (34.8) 1.5418 6 100 P41212 87.0, 103.7 3.1 39,113 7555 5.2 99.0 (99.9) 15.2 (2.5) 3.6 (30.6) 1.0053 6 100 P41212 86.9, 104.2 2.3 174,872 18,027 9.7 98.8 (99.6) 6.2 (2.2) 6.6 (34.7)

R-Axis IV⫹⫹

R-Axis IV⫹⫹

100 APS, GMCA-CAT, 23 ID-D Mar CCD 300 100 Rigaku RU-H3RHB 100 Rigaku RU-H3RHB

100 APS, GMCA-CAT, 23 ID-D Mar CCD 300

100 APS, IMCA, 17 ID-D ADSC Mar CCD 165 1.0000 NA NA P41212 86.6, 104.3 1.85 157,823 34,235 4.6 99.2 (100) 12.6 (2.2) 3.3 (35.3) 100 APS, IMCA, 17 ID-D ADSC Mar CCD 165 0.9641 NA NA P41212 86.3, 106.2 2.3 103,091 18,352 5.6 99.7 (100) 10.0 (2.1) 5.0 (37.2) 100 APS, IMCA, 17 ID-D ADSC Mar CCD 165 0.9794 NA NA P41212 86.3, 106.2 2.3 103,129 18,340 5.6 99.6 (100) 9.1 (2.6) 5.4 (29.8) Data set Temperature (K) X-ray source

MAP2569c䡠UDP䡠Glc (pH 5.5) MAP2569c䡠Mn2ⴙ䡠UDP-Glc (pH 7.0) MAP2569c䡠Mn2ⴙ䡠UDP (pH 7.0) MAP2569c䡠citrate (pH 5.5) Remote Inflection Peak

APS, Advanced Photon Source (Chicago, IL); NA, not available.

TABLE 1 Data collection and phasing statistics

100 APS, IMCA, 17 ID-D ADSC Mar CCD 165 0.9793 NA NA P41212 86.3, 106.2 2.3 103,153 18,330 5.7 99.6 (100)a 9.7 (2.9) 5.0 (26.0)

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Crystal Structure of MAP2569c—Although Rv1208 was insoluble, the expression of its close homologue, MAP2569c, yielded soluble protein that was amenable for structural studies (10). We subsequently determined the crystal structure of apo-MAP2569c using the MAD technique (see “Experimental Procedures” and Table 1). The structure was refined 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 and B). At the core of MAP2569c is a mixed nine-stranded twisted ␤-sheet (sA) with topology ␤5-␤4-␤3-␤6-␤8-␤7-␤9-␤2-␤1 (␤1 and ␤7 are antiparallel), flanked by three helical segments on either side. As expected from its low sequence similarity to the GT2 family, MAP2569c contained the GT-A scaffold observed in SpsA (8) (see Fig. 1, A and B) and can be subdivided into an N-terminal domain, “catalytic” domain, and C-terminal domain. The catalytic domain of MAP2569c (residues 50 –260) exhibited the characteristic ␣/␤/␣ architecture of the GT-A fold, which is subdivided into “nucleotide-binding” (residues 50 –138) and “acceptor-binding” (residues 142–260) subdomains. The nucleotide-binding subdomain of MAP2569c comprised 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 disordered region (residues 174 –188). The signature catalytic DXD motif of the GT-A fold is found, as expected, at the juncture of the two catalytic subdomains and comprised Asp-139 –Ser140 –Asp-141. A second small antiparallel ␤-sheet (sB), perpendicular to the central ␤-sheet (sA), is formed by two short ␤-stands: ␤6⬘ at the N terminus of the acceptor-binding subdomain and ␤9⬘ within a short loop bridging the acceptor-binding subdomain and C-terminal domain. This loop (residues 261– 267) sat adjacent to the DXD motif and traced the nucleotidebinding/acceptor-binding subdomain divide. The C-terminal domain (residues 268 –329) contained a long helical region and a 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 unusual C-terminal tail wrapped around its 2-fold related partner (see Fig. 1C). The remainder of this dimeric interface is formed by residues in the long helical region at the start of the C-terminal domain and in the N-terminal domain and acceptor-binding subdomain. The buried surface area at this dimeric interface was quite extensive (⬃1900 Å2) and is therefore likely to represent a biological dimer. Supporting this assertion are AUC studies of MAP2569c, which was shown to be a dimer in aqueous solution (see supplemental Fig. 1 and supplemental Table 1). Given the location of the substrate-binding sites, it appears that the MAP2569c dimer is probably required for stability as opposed to catalysis. Accordingly, the dimeric MAP2569c exhibits a GT-A fold and DXD motif consistent with GT activity.

MAP2569c䡠UDP䡠GlcNAc (pH 7.0)

RESULTS

Crystal Structure of a Mycobacterial Glycosyltransferase of the GT-A Fold TABLE 2 Refinement statistics Model Nonhydrogen atoms Protein Ligand/ion Water Resolution range (Å) Rfactor/Rfreea (%) Root mean square deviation from ideal values Bond lengths (Å) Bond angles (degrees)

a

MAP2569c䡠Citrate MAP2569c䡠Mn2ⴙ䡠UDP MAP2569c䡠Mn2ⴙ䡠UDP-Glc MAP2569c䡠UDP-Glc MAP2569c䡠UDP-GlcNAc (pH 7.0) (pH 7.0) (pH 5.5) (pH 7.0) (pH 5.5) 2289 42 102 30–1.85 18.4/19.8

2255 31 36 30–2.3 19.3/21.7

2252 42 4 30–3.1 19.6/23.4

2289 38 24 30–2.6 18.0/21.9

2294 38 48 30–2.2 18.8/20.5

0.015 1.452

0.016 1.505

0.016 1.298

0.015 1.622

0.015 1.414

Ramachandran plot (%) Most favored regions Additional allowed regions

91.5 8.5

91.5 8.5

89.8 9.4

90.2 9.4

91.9 8.1

B-factors (Å2) Average main chain Average side chain Average ligand/ion Average water molecule

27.0 28.9 61.4 42.5

45.5 46.3 61.4 46.4

42.0 42.4 88.1 53.2

29.8 31.0 42.5 42.4

44.7 45.6 65.2 42.8

Rfactor ⫽ ⌺hkl储FP(obs)兩 ⫺ 兩FP(cal)储/⌺hkl兩FP(obs)兩 for all reflections. For refinement of the apo crystal form, Rfree was calculated using randomly selected reflections (10%). For refinement of the ligated MAP2569c complexes, the same Rfree data set was used as selected in the apo crystal form.

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applied with other GTs to identify the donor sugar (25). Namely, we soaked 10 mM CMP, GDP, and UDP nucleotides into the apocrystal form. The difference Fourier electron density maps calculated using these data (statistics not shown) revealed evidence for UDP only and not for CMP or GDP at the predicted donor substrate site of MAP2569c and thus indicated that UDP is the likely nucleotide component of the donor substrate for MAP2569c. We subsequently determined the 2.3 Å resolution structure of MAP2569c in complex with its donor substrate product, Mn2⫹䡠UDP, at pH 7.0 and refined it to Rfactor and Rfree values of 19.3 and 21.7%, respectively (see “Experimental Procedures”) (Tables 1 and 2 and Fig. 2A). Overall, the structure of MAP2569c is unchanged on binding UDP, although a major conformational change was observed in the loop (residues 261–267) linking the acceptor-binding subdomain and C-terminal domain (see below). The ␣- and ␤-phosphates on UDP interact with Asp-141O␦2 of the DXD motif via the Mn2⫹ ion (Fig. 3A), as observed previously in the SpsA structure (6). In addition, His-263N␦1 on the loop linking the acceptor-binding subdomain and C-terminal domain also coordinates the Mn2⫹ ion. The ␣-phosphate also hydrogen-bonds to Tyr-234O␩, and the ␤-phosphate forms a water-mediated hydrogen bond with Asp-139O␦2 and is further within van der Waals contact of the side chains of Met-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 van der Waals contacts with the side chains of Pro-55 and Ser-86. The O2 on the uracil hydrogen-bonds to one of the two alternate conformations modeled for Ser-86O␥. In addition to these contacts with the nucleotide-binding subdomain, the uracil is also in van der Waals 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, and Tyr-234. The O2* on the ribose hydrogen-bonds to Leu-57N and the carboxylates of Glu59, and the O3* hydrogen-bonds to Pro-55O. The ribose ring further interacts with the conserved DXD motif, with the O3* also hydrogen-bonding to the Ser-140N,O␥. Thus, the large number of contacts between MAP2569c and UDP provided a basis for its specificity for this nucleotide. VOLUME 283 • NUMBER 41 • OCTOBER 10, 2008

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Comparison of the GT-A Fold of MAP2569c with Other Glycosyltransferases—We next compared the GT-A fold of MAP2569c to other structures in the PDB using the DALI server (21) and identified a number of nucleotide-binding proteins with similar architecture. The closest structural match (with a Z-score of 18.6) was to the catalytic domain of mannosylglycerate synthase (MGS) from Rhodothermus marinus (22) (Protein Data Bank code 2bo4, 181 C␣ atoms; root mean square deviation of 2.0 Å, and 22% sequence identity). Based on sequence similarity, MGS was previously classified as a member of the GT2 family (23); however, MGS was subsequently shown to use a retaining mechanism and was reclassified as the founding member of the GT78 family (22). The next two closest structural homologues from the GT superfamily (with Z-scores of 13.5 and 12.4, respectively) belong to SpsA from B. subtilis (8) (Protein Data Bank code 1qg8, 166 C␣ atoms, root mean square deviation of 3.4 Å and 13% sequence identity) and polypeptide N-acetylgalactosaminyltransferases from Homo sapiens (24) (Protein Data Bank code 2ffu, 171 C␣ atoms, root mean square deviation of 3.0 Å, and 16% sequence identity). As with MGS, polypeptide N-acetylgalactosaminyltransferase was initially grouped with SpsA in the GT2 family but has since been shown to use a retaining mechanism and was subsequently reclassified as a member of the GT27 family (19, 21). Of the 20 highest structural matches to the GT-A fold of MAP2569c, only six are GTs. Consequently, the GT-A fold of MAP2569c shows highest structural homology to the catalytic domain of a GT with a retaining mechanism and displays significant structural homology to other nucleotide-binding protein families. This structural similarity of MAP2569c to other nucleotide-binding protein families is indicative of both the structural diversity of and lack 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 nucleotidesugar as its activated donor substrate (23). Accordingly, we sought to define the nucleotide specificity of this enzyme. First, we screened for nucleotide binding via a crystallographic approach. A crystallography-based approach has previously been

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

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using these UDP-sugars, there was electron density for at least the uracil ring of the UDP-sugar at the predicted donor substrate site of MAP2569c. For example, the complex with UDP-GlcNAc was refined to 2.2 Å to Rfactor and Rfree values of 18.8 and 20.5%, respectively (see “Experimental Procedures” and Tables 1 and 2) and revealed evidence for the uracil and ribose rings and ␣-phosphate of the UDP-sugar (see supplemental Fig. 2). However, only in the complex with UDP-Glc was electron density also observed for Mn2⫹, the ␤-phosphate, and the sugar moiety (see below) (Fig. 2B) at the predicted donor substrate site. This suggests specificity for UDPGlc over all other natural, including chemically related, UDP-sugars. Second, to further verify this specificity, the enzyme activity of MAP2569c was analyzed with the UDP-sugars found in mycobacteria, UDP-GlcNAc, UDP-Gal, and UDPGlc. The highest activity was observed with UDP-Glc, with no activity toward UDP-Gal or UDPGlcNAc (Fig. 3), suggesting that MAP2569c uses UDP-Glc as its natural donor sugar. There was some residual activity observed with GDP-Glc (20% of that observed with UDP-Glc), suggesting that there is some plasticity in donor sugar binding (Fig. 3). UDP-Glc sat in a “folded back” conformation at the putative donor site, reminiscent of the conformation first observed in a donor substrate analogue in complex with the FIGURE 1. Ribbon representations of the structure of MAP2569c. MAP2569c is shown in the same orienta2⫹ tion in complex with citrate (A) and Mn 䡠UDP-Glc (B) and colored according to domain or subdomain. Disor- retaining GT LgtC (26), with a burdered regions are indicated by dashed lines. Secondary structure was calculated using DSSP (34). The N-termi- ied surface area of ⬃50 Å2. The nal domain is colored pink, the Rossmann domain is shown in cyan, the catalytic DXD motif is shown in red, the donor substrate traverses the cleft acceptor-binding domain is shown in green, the flexible loop is shown in yellow, the long helical region is in purple, and the remainder of the C-terminal domain (residues 286 –329) is shown in salmon. The ligands are between the nucleotide- and shown in a ball and stick representation, with citrate superimposed in the complex with Mn2⫹䡠UDP-Glc. C, the acceptor-binding subdomains (see dimer. The 2-fold symmetry-related molecules are shown in pink and yellow. Disordered regions are indicated Fig. 1B). The interactions with by dashed lines. This figure and all figures in this paper were drawn using PyMOL (35). UDP are similar to those observed MAP2569c Complexed with UDP-glucose—To identify what in the complex of MAP2569c with Mn2⫹䡠UDP. The Glc moiety of the donor substrate is within hydrogen UDP-sugar MAP2569c could bind, a similar co-crystallization approach was employed. UDP-activated sugars that act as bonding distance of several residues within MAP2569c (see natural donor substrates for GTs include UDP-GalNAc, UDP- below) (Figs. 2C and 4). The O4⬘ and O6⬘ OH moieties hydroGlcNAc, UDP-␣-L-arabinose, UDP-Gal, UDP-Glc, UDP-␣-D- gen-bond to Glu-237O⑀1 on the ␣-helix hP; the O3⬘-OH and glucuronic acid, and UDP-␣-D-xylose (although only UDP- O4⬘-OH hydrogen-bond to Lys-119N␨, and the O3⬘-OH further GlcNAc, UDP-Gal, and UDP-Glc have been found in hydrogen-bonds to Asp-139O␦2. In addition, Asp-139O␦2 forms mycobacteria, all natural UDP-sugar donor substrates were a water-mediated hydrogen bond with the O2⬘-OH. The investigated for comparison). For all crystal structures solved O5⬘-OH hydrogen-bonds to Leu-214O, and the O6⬘-OH fur-

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

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ligated forms. The comparative analyses revealed that upon ligation, the loop (residues 261–267) linking the catalytic and C-terminal domains in MAP2569c changes conformation. In the apo form of MAP2569c, residues 262–263 on the flexible loop form a short ␤-strand (␤9⬘) of the “transient” ␤-sheet (sB, ␤6⬘-␤9⬘) and residues 268 –270 form a 310helix (hO) at the start of the C-terminal domain (see Fig. 5). However, in the MAP2569c䡠Mn2⫹䡠 UDP-Glc complex, this flexible loop adopted a different conformation, driven by the participation of the imidazole group on His-263 in a Mn2⫹-mediated interaction with the ␣- and ␤-phosphates on UDPGlc (see Fig. 5). To accommodate this, the ␤9⬘ and ␤6⬘ strands are restructured into loops, and residues 268 –270 form an ␣-helix. In the complex of MAP2569c䡠 Mn2⫹䡠UDP, the loop adopted an “intermediate” conformation (root mean square deviation of ⬃0.4 Å) to that observed in the apo and “donor substrate” liganded forms of MAP2569c. Moreover, the ␤-phosphate on UDP interacted less with the C-terminal domain than in the complex with Mn2⫹䡠UDP-Glc. FIGURE 2. Unbiased electron density for the putative donor substrate product and donor substrate of Instead, the ␤-phosphate made van MAP2569c. A, Mn2⫹䡠UDP; B, Mn2⫹䡠UDP-Glc at pH 7.0; C, UDP-Glc at pH 5.5. A–C, the electron density maps der Waals contacts with the side shown are Fo ⫺ Fc simulated annealing omit maps contoured at 3␴. The Mn2⫹ ion and water molecules are represented by purple and red spheres, respectively. Hydrogen bonds and Mn2⫹-mediated interactions are chain of Arg-266 (disordered in the represented by dashed black and purple lines, respectively. apo and Mn2⫹䡠UDP-Glc liganded forms of MAP2569c) and is within water-mediated hydrogen bondther forms water-mediated contacts with Tyr-234O and ing distance of Asp-139O␦2. Consequently, the conformaIle-238N. Given that the O4⬘-OH is the only distinction tional changes observed in the flexible loop containing Hisbetween UDP-Glc and UDP-Gal, the interactions with this oxy- 263 and associated changes in adjacent secondary structural gen are probably important for discriminating the natural elements are important for coordination of the donor subdonor substrate for MAP2569c. The C6⬘ and O6⬘ make van der strate and for stabilization of the donor substrate product of Waals contacts with the side chain of Leu-214, and the C5⬘ and MAP2569c. The Role of His-263—To further investigate the involvement O5⬘ also make van der Waals contacts with the side chain of Met-274, which adopts a different conformation to accommo- of His-263 in Mn2⫹ coordination, we determined the crystal date the Glc moiety. Consequently, the sugar moiety of the structure of MAP2569c in complex with Mn2⫹ and UDP-Glc at donor substrate for MAP2569c interacts with residues from the a pH value below the pKa of histidine (pH 5.5 was used in this catalytic and C-terminal domains of MAP2569c, and the large study). At such a pH value, histidine acts as an acid rather than number of contacts dictates the specificity for UDP-Glc. as a base (as under physiological conditions) and thus cannot Induced Fit—The crystal structures of GTs of the GT-A participate in Mn2⫹ coordination. The complex with fold have often indicated that a flexible loop in the vicinity of Mn2⫹䡠UDP-Glc at pH 5.5 was refined to 2.6 Å to Rfactor and Rfree the nucleotide-binding site plays a critical role in the cata- values of 18.0 and 21.9%, respectively (see “Experimental Prolytic mechanism of the enzyme (for a review, see Qasba et al. cedures”) (Tables 1 and 2). The structure revealed evidence for (27)). To investigate whether induced fit plays a role in the uracil and ribose rings and ␣-phosphate of the UDP-sugar MAP2569c, we compared the structures of the apo and only (see Fig. 2C), indicating the requirement for the ionizable

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

FIGURE 5. Comparison of the flexible loop of MAP2569c in complex with citrate, Mn2ⴙ䡠UDP, and Mn2ⴙ䡠UDP-Glc at pH 7.0. The shift in the flexible loop containing His-263 (shown in a ball and stick representation) and associated conformational changes in adjacent secondary structural elements between MAP2569c in complex with citrate (in salmon), Mn2⫹䡠UDP (in cyan), and Mn2⫹䡠UDP-Glc at pH 7.0 (in green) reshape the “active site” of MAP2569c.

FIGURE 4. Comparison of the donor substrate binding site of MAP2569c to that found in MGS. MGS (pink) in complex with Mn2⫹䡠GDP-Man (donor substrate not shown) superposed on MAP2569c (green) in complex with Mn2⫹䡠UDP-Glc. The hydrogen-bonding network spanning Glu-237 to Asp139 is represented by dashed black lines. Other hydrogen bonds and Mn2⫹mediated interactions observed in the MAP2569c䡠Mn2⫹䡠UDP-Glc complex at pH 7.0 are represented by dashed gray and purple lines, respectively.

imidazole group on His-263 for Mn2⫹ coordination, and thus also for ␤-phosphate and sugar coordination, in the complex of MAP2569c with Mn2⫹䡠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 donor substrate, the catalytic domain of MAP2569c showed the highest structural homology to N-acetylglucosaminyltransferase I from Oryctolagus cuniculus (28) (Z-score 12.0, Protein Data Bank code 1foa, 164 C␣ atoms superimpose with a root mean square deviation of 3.2 Å and a sequence identity of 13%) (21). Although the flexible loop in N-acetylglucosaminyltransferase I also undergoes conformational change on activated donor substrate coordination, no residue on this loop is directly involved in divalent metal coordination. In addition, rather than trace OCTOBER 10, 2008 • VOLUME 283 • NUMBER 41

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 of the GT-A fold and identified structurally homologous segments in the “retaining” GTs MGS (22) and polypeptide N-acetylgalactosaminyltransferase (24). In all three structures, a histidine (as part of an Asp/His motif) within this loop is required for divalent metal ion coordination. In addition, there is a similar long helical region at the start of the C-terminal domain. In MAP2569c and MGS, structurally homologous methionine side chains in this region (Met-274 in MAP2569c and Met-229 in MGS) have also been shown (here) to play analogous 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 in complex with MGS, and the mode of sugar coordination in these structures is similar (see Fig. 4) The side chains of Leu163, Trp-189, and Met-229 in MGS overlay with those of Leu214, Tyr-234, and Met-274 in MAP2569c and similarly form hydrophobic contacts with the sugar moiety in these structures. The side chains of Asp-192 in MGS and Glu-237 in MAP2569c are both within hydrogen bonding distance of the O4⬘ and O6⬘ JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 3. NDP-sugar specificity for MAP2569c. Relative activity of MAP2569c in the presence of different donor NDP-sugars and the acceptor molecule, 3-phosphoglycerate (33). The results are expressed as relative activity of the highest value and are the mean ⫾ S.D. of three experiments performed in triplicate.

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

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However, additional residues on the flexible loop in MGS, which is notably partially disordered in the complex of MGS with Co2⫹䡠GDP, participate in Mn2⫹䡠GDP-Man coordination, with Arg-218 and Tyr-220 also interacting with the ␣- and ␤-phosphates on GDPMan, respectively (see Fig. 6B). The structural homology observed between MAP2569c and retaining GTs of the GT-A fold extends beyond the catalytic domains of these enzymes to the flexible loops and adjacent secondary structural elements that undergo significant conformational change on donor substrate coordination. FIGURE 6. Comparison of the flexible loop of MAP2569c in complex with its putative donor substrate and The Acceptor Binding Site of those of inverting and retaining GTs of the GT-A fold. A, N-acetylglucosaminyltransferase I (in purple) in complex with Mn2⫹䡠UDP-GlcNAc (donor substrate not shown) superposed on MAP2569c (in green) in complex MAP2569c—A bound organic molwith Mn2⫹䡠UDP-Glc (shown in a ball and stick representation). B, MGS (in pink) in complex with Mn2⫹䡠GDP-Man ecule often indicates an important (donor substrate not shown) superposed on MAP2569c (green) in complex with Mn2⫹䡠UDP-Glc (shown in a ball (functional) site in a crystal strucand stick representation). The Mn2⫹-mediated interactions between the N␦1 on His-263 of MAP2569c and the ture. Indeed, the binding site of a ␣- and ␤-phosphates on UDP-Glc are represented by dashed purple lines. citrate molecule in the apo form of MAP2569c is similarly located to the binding site observed for a citrate in the apo form of MGS (see Fig. 7, A and B), with Thr-192 and Arg261 in MAP2569c and Thr-139 and Arg-131 in MGS playing analogous hydrogen-bonding roles in coordinating the citrate in these structures. Indeed, the citrate in MGS notably makes interactions with MGS similar to that of the natural acceptor substrate of MGS, D-glycerate, indicating the adaptability of this binding site. Although the threonines within hydrogen bondFIGURE 7. Comparison of the citrate binding site of MAP2569c to that found in MGS. A, MAP2569c; B, MGS. ing distance of the citrate are from For both structures, the Rossmann subdomain is shown in cyan, the catalytic DXD motif is shown in red, the structurally homologous segments acceptor-binding subdomain is shown in green, the flexible loop is shown in yellow, the structurally homologous helical region at the start of the C-terminal domain is shown in purple, and the remainder of the C-terminal of MAP2569c and MGS, the argidomain is shown in salmon. The residues with side chains involved in direct hydrogen-bonding interactions nine residues are from different with the bound solvent molecule are also shown in ball and stick representations. The electron density shown regions. To illustrate, Arg-261 in for citrate is a final 2Fo ⫺ Fc synthesis contoured at 1␴. In MGS, Thr-139 and Arg-131 also form direct hydrogen MAP2569c is from the flexible loop bond interactions with the natural acceptor substrate D-glycerate. involved in “substrate donor” coordination, whereas Arg-131 in MGS on the sugar. In addition, Glu-237 in MAP2569c is involved in a is from a loop that structurally corresponds to the disordered hydrogen bonding network via Lys-119 that extends to the first region (residues 174 –188) in MAP2569c. Consequently, aspartate of the DXD motif. This network, also observed in the although the citrate binding sites in the apo form of MAP2569c structures of the retaining GTs LgtC (26) and MGS (22) in com- and MGS are similarly located, the arginines within hydrogen plex with Mn2⫹䡠UDP-2-deoxy-2-fluoro-Gal (a donor substrate bonding of the citrate in MAP2569c and MGS are from nonanalogue) and Mn2⫹䡠GDP-Man, respectively, involves the basic structurally homologous segments of the polypeptide chain, and acidic side chains of Lys-119 (Lys-76 in MGS) and Asp-139 suggesting that the mode of natural acceptor substrate binding (Asp-100 in MGS), both of which are within hydrogen bonding may differ between MAP2569c and MGS. distance of the O3⬘ (see Figs. 3B and 4). The main chain carbonSequence Similarity of MAP2569c to Putative Orthologues yls on Leu-163 in MGS and Leu-214 also participate in analo- from the Corynebacterineae—To gain further insight into the function of MAP2569c, we compared the sequence of gous hydrogen-bonding interactions with the sugar moiety.

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

DISCUSSION The ubiquitous nature of oligosaccharides and glycoconjugates and the rapidly growing number of candidate GT sequences uncovered by genomic analyses are indicative of a wealth of untapped knowledge on carbohydrate function. Due to the distant sequence similarities between some GT families, crystal structure analysis, as exemplified here, presents a powerful tool for establishing the relatedness between GTs and helps to bridge the chasm between the vast amount of sequence data and the paucity of functional and mechanistic knowledge about this important class of enzymes. We have determined the structure of MAP2569c, which possessed a GT-A fold and a DXD motif associated with GT2 family activity. MAP2569c contains a flexible loop, which undergoes conformational change on activated donor substrate binding and houses a histidine residue required for divalent metal ion coordination, as has also been observed in the inverting GT, ␤4-galactosyltransferase (30). However, the overall fold and catalytic core of MAP2569c exhibits highest structural similarity to MGS, a GT that has been shown to use a retaining mechanism and represents the archetype of the GT78 family (22). Moreover, the structural homologies observed between MAP2569c and retaining GTs of the GT-A fold extend beyond the flexible loop in the vicinity of the activated donor substrate binding site to the adjacent secondary structural elements that also undergo conformational change on activated donor substrate coordination. Through crystallographic and enzymatic analyses, we revealed MAP2569c preferentially binds the activated donor substrate Mn2⫹䡠UDP-Glc. The interactions between MAP2569c and Mn2⫹䡠UDP-Glc are also reminiscent of those observed between MGS and its activated donor subOCTOBER 10, 2008 • VOLUME 283 • NUMBER 41

strate (22). This level of specificity of MAP2569c is rather unusual in comparison with the GTs in general. GTs use a carboxylate residue to bind the acceptor substrate and initiate transfer of the sugar moiety (27). The acidic residue in the retaining GT MGS (Asp-192) is structurally conserved in MAP2569c (Glu-237). Moreover, these residues are involved in similar hydrogen-bonding interactions at the donor substrate sites of MAP2569c and MGS. Nevertheless, as noted by Flint et al. (22), the acidic residue is similarly located in GTs of the GT-A fold that use both inverting and retaining mechanisms, possibly reflecting the evolution of retaining GTs of the GT-A fold from the inverting GT2 family (31). Flint et al. (22) also suggested that a change in the angle of the corresponding ␣-helices containing the acidic group in inverting versus retaining GTs of the GT-A fold could define the mechanism of these enzymes. However, when comparing MAP2569c and other GTs, we could find no such correlation (data not shown). Rather, one shared feature observed in retaining GTs of the GT-A fold is the position of the side chain of the acidic residue on the nucleoside-proximal side of the sugar moiety, as opposed to the nucleoside distal side of the sugar moiety, as observed in inverting GTs of the GT-A fold (see supplemental Fig. 4) (32). Although there are no clear structural features that define the mechanism of GTs, based on the accumulative structural homologies between MAP2569c and MGS, we suggest that MAP2569c possesses the same retaining mechanism of MGS, but this contention will require further experimentation. We also provide evidence that the orthologues of MAP2569c from other Mycobacterium sp. and from related Corynebacterineae share a common architecture. Moreover, given that the residues involved in donor and acceptor substrate recognition are also conserved across the orthologues, we suggest that these also exhibit similar substrate specificities and hence perform a similar role in the biosynthesis of the unique cell wall of these bacteria. Although all known and characterized GTs of the GT-A fold found in Mycobacterium species are members of the inverting GT2 family (3), our analyses indicate that some GTs may need to be reclassified. A recent report describes glycosyl-3-phosphoglycerate synthase from Mycobacterium smegmatis and Mycobacterium bovis that has homology (⬃25% sequence identity) to the M. tuberculosis H37Rv gene Rv1208. Glucosyl-(1–2)-glycerate is found at the reducing end of methylglucose lipopolysaccharide, which is involved in regulating fatty acid synthesis in Mycobacterium (33). The formation of glucosylglycerate is performed by glycosyl-3-phosphoglycerate synthase, in which it transfers glucose from NDP-glucose to glucosyl-3phophoglycerate. Recombinant glycosyl-3-phosphoglycerate synthase from M. bovis showed optimal activity with UDP-Glc and strictly required Mg2⫹. Glycosyl-3-phosphoglycerate synthase enzymes are classified into the retaining family 81 of GTs. These observations lend support to MAP2569c (and Rv1208) being a UDPGlc specific GT that uses a retaining mechanism. Given the unique complex carbohydrate content of the Mycobacterium cell wall, GTs involved in its biosynthesis present promising targets for new drugs in the treatment of tuberculosis and other diseases caused by Mycobacterium species. Previous studies using saturation mutagenesis screening have JOURNAL OF BIOLOGICAL CHEMISTRY

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MAP2569c with those of its putative orthologues from closely related Mycobacterium species, including Mycobacterium ulcerans, M. tuberculosis, and Mycobacterium leprae, and from other representatives of the Corynebacterineae, including Nocardia farcinica and Corynebacterium glutamicum (see supplemental Fig. 3). The sequence identity to MAP2569c, calculated using ClustalW (29), of these orthologues ranges from 47 to 85%, with a high level of identity for all sequences throughout the catalytic domain (residues 50 –260), flexible loop (residues 261–267), and long helical region at the start of the C-terminal domain (residues 268 –285) of MAP2569c, suggesting that these orthologues from the Corynebacterineae share the same architecture for these structural modules involved in donor substrate 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 der Waals interactions with UDP-Glc at pH 7.0 are conserved in these orthologues. Moreover, the citrate-binding site in MAP2569c is well conserved throughout the orthologues. This essentially invariant nature of the donor and potential acceptor substrate binding sites in MAP2569c across the orthologues from the Corynebacterineae suggests that each of these candidate GTs performs a similar role in the synthesis of the unique cell wall of this suborder.

Crystal Structure of a Mycobacterial Glycosyltransferase of the GT-A Fold suggested that the M. tuberculosis homolog Rv1208 is essential for survival and growth (9). The function of Rv1208 is not yet known, but its most likely role is as a putative GT in Mycobacterium cell wall biosynthesis. Its high specificity for its candidate activate donor substrate together with a natural acceptor substrate that is probably exclusively found in Corynebacterineae makes the orthologues of MAP2569c promising targets for new antimicrobials. Acknowledgments—We thank the IMCA and GM/CA-CAT staff for assistance in data collection at the Advanced Photon Source (Chicago, IL). REFERENCES

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1. Crick, D. C., Mahapatra, S., and Brennan, P. J. (2001) Glycobiology 11, 107R–118R 2. Daffe, M., and Draper, P. (1998) Adv. Microb. Physiol. 39, 131–203 3. Berg, S., Kaur, D., Jackson, M., and Brennan, P. J. (2007) Glycobiology 17, 35R–56R 4. Wimmerova, M., Engelsen, S. B., Bettler, E., Breton, C., and Imberty, A. (2003) Biochimie (Paris) 85, 691–700 5. Rossmann, M. G., Moras, D., and Olsen, K. W. (1974) Nature 250, 194 –199 6. Wiggins, C. A., and Munro, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7945–7950 7. Martinez-Fleites, C., Proctor, M., Roberts, S., Bolam, D. N., Gilbert, H. J., and Davies, G. J. (2006) Chem. Biol. 13, 1143–1152 8. Charnock, S. J., and Davies, G. J. (1999) Biochemistry 38, 6380 – 6385 9. Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2003) Mol. Microbiol. 48, 77– 84 10. Fulton, Z., Crellin, P. K., Brammananth, R., Zaker-Tabrizi, L., Coppel, R. L., Rossjohn, J., and Beddoe, T. (2008) Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 428 – 431 11. Studier, F. W. (2005) Protein Expression Purif. 41, 207–234 12. Ccp4. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760 –763 13. Weeks, C. M., Blessing, R. H., Miller, R., Mungee, R., Potter, S. A., and Rappleye, J., Smith, G. D., Xu, H., and Furey, W. (2002) Z. Kristallogr. 217, 686 – 693 14. Cowtan, K. (1994) Joint CCP4 and ESF-EACBM Newsletter on Protein

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SUPPLEMENTAL MATERIAL FIGURE 1. Sedimentation velocity analysis of MAP2569c. A, the absorbance at 295 nm is plotted as a function of radial position from the axis of rotation (cm) for MAP2569c (1.5 mg/ml) at 6 min intervals. The raw data is plotted as open symbols and the solid lines represent the nonlinear least squares best fits to the c(M) distribution shown in B. B, continuous mass [c(M)] distribution is plotted as a function of molecular mass (kDa) for MAP2569c. The c(M) distribution was calculated using P = 0.95 with 200 molecular masses ranging from 0.3 kDa to 200 kDa. The distribution shows a single species with molecular mass of ∼71.7 kDa, which is in excellent agreement with the theoretical molecular mass of a MAP2569c dimer. The hydrodynamic properties of the MAP2569c dimer are summarized in Supplemental Table 1. Inset, Residuals for c(M) distribution best-fit shown plotted as a function of radial position (cm). FIGURE 2. Unbiased electron density for UDP-GlcNAc in complex with MAP2569c at pH 7.0. The electron density map shown is a Fo - Fc simulated annealing omit map contoured at 3σ. Hydrogen bonds are represented by dashed black lines. FIGURE 3. Multiple amino acid sequence alignment of MAP2569c from M. avium subsp. paratuberculosis and its putative orthologues from M. ulcerans, M. tuberculosis, M. leprae, Nocardia farcinica, and Corynebacterium glutamicum. The numbering is for MAP2569c from M. avium. β-strands and α- and 310-helices calculated using DSSP (32) are indicated by green arrows and purple and pink zig-zag lines above the sequence alignment, respectively. Disordered regions in the crystal structure of MAP2569c are indicated by grey lines. The sequences were obtained using the program PSI-BLAST (34) and the alignment was performed with ClustalW (29). Conserved residues are highlighted in yellow and conservatively substituted residues are shown in blue. MAP2569c residues involved in (direct or indirect) hydrogen bond, Mn2+–mediated, or van der Waals interactions with UDP-Glc at pH 7.0 are shown in red in bold. MAP2569c residues with side chains involved in direct hydrogen bond, Mn2+–mediated, or van der Waals interactions with UDP-Glc at pH 7.0 are also boxed. MAP2569c

residues involved in hydrogen bond or van der Waals interactions with citrate at pH 5.5 are shown in blue in bold. MAP2569c residues with sidechains involved in hydrogen bond or van der Waals interactions with citrate at pH 5.5 are also boxed. (Asp-139, Leu214 and His-263 interact with Mn2+⋅UDP-Glc at pH 7.0 and with citrate at pH 5.5). a The pairwise score (34,35) represents the sequence identity to MAP2569c and was calculated using ClustalW (29). b M. avium represents M. avium subsp. paratuberculosis. FIGURE 4. Comparison of the predicted catalytic base in MAP2569c in complex with its putative donor substrate to those of retaining and inverting GTs of the GTA fold. MGS (pink) and GnT I (purple) in complex with Mn2+⋅GDP-Man and Mn2+⋅UDPGlcNAc, respectively (activated donor substrates not shown), superposed on MAP2569c (green) in complex with Mn2+⋅UDP-Glc. The side chains of the acidic residue in these complexes (Glu-237 in MAP2569c, Asp-192 in MGS, and Asp-291 in GnT I) are shown in ball and stick. In MAP2569c and the retaining MGS, the side chain is on the nucleoside-proximal side of the sugar moiety. In the inverting GnT I, the side chain is on the nucleoside-distal side of the sugar moiety.

SUPPLEMENTAL TABLE 1

Hydrodynamic properties of MAP2569c measured by sedimentation velocity analysis Mr 34,374.2

M1

s20,w

(kDa)

(S)

71.7

4.45

f/f01

Dimensions2

a/b2

1.36

139Å × 42.2Å

3.28

1

Taken from the c(M) distribution best-fit (supplemental Fig. 1).

2

Calculated assuming a prolate ellipsoid and hydration of 0.397 g/g using the Vbar

method (18).

SUPPLEMENTAL FIGURE 1

A

B

SUPPLEMENTAL FIGURE 2

SUPPLEMENTAL FIGURE 3

SUPPLEMENTAL FIGURE 4

7

Crystal Structure of a UDP-glucose-specific Glycosyltransferase from a Mycobacterium Species 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 and Travis Beddoe J. Biol. Chem. 2008, 283:27881-27890. doi: 10.1074/jbc.M801853200 originally published online July 30, 2008

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