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J. Biol. Chem., Vol. 279, Issue 31, 32684-32691, July 30, 2004

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The Position of a Key Tyrosine in dTDP-4-Keto-6-deoxy-D-glucose-5-epimerase (EvaD) Alters the Substrate Profile for This RmlC-like Enzyme^*

Alexandra B. Merkel, Louise L. Major, James C. Errey, Michael D. Burkart¶||, Robert A. Field, Christopher T. Walsh¶, and James H. Naismith**

From the Centre for Biomolecular Sciences, The University, St. Andrews, Scotland, KY16 9ST, United Kingdom, the Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, Norfolk, NR4 7TJ, United Kingdom, and the ^¶Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, April 13, 2004 , and in revised form, May 24, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 Å structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction (double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Glycopeptide antibiotics are highly effective clinical agents used to combat bacterial infections caused by many drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (1, 2). In the glycopeptide superfamily, vancomycin is one of the most effective antibiotic compounds, and its use has increased dramatically over the past decade (2, 3). However, the emergence of vancomycin-resistant bacteria, especially enterococci, has increased the need for novel therapeutic agents. Vancomycin inhibits bacterial cell wall synthesis by interfering with the polymerization of the phosphodisaccharide-pentapeptide lipid complex. It binds to the free C termini of D-alanyl-D-alanine-containing peptides, preventing their use as transpeptidase substrates (4). Thus, in the presence of vancomycin peptidoglycan cross-linking is blocked, and membrane-bound lipid intermediates accumulate (5).

All vancomycin-type antibiotics consist of an identical heptapeptide backbone with glucose attached to the oxygen of the tyrosine side chain of residue 4 of the aglycone. One or more sites of the glycopeptide can then be further glycosylated (6), often with unusual deoxy-sugars, creating a wide diversity of structures. The carbohydrate chains are required for biological activity (7-9), and recent studies have shown that the disaccharide is bactericidal in its own right (10, 11).

Although its total chemical synthesis has been reported (12, 13), vancomycin for therapeutic use is still produced by fermentation. Manufacture of novel analogues to overcome or manage resistance will therefore most likely arise via synthetic modification of the fermented product or genetic engineering of the organism to produce altered products. The latter route is particularly attractive because it may allow considerable structural diversity and will utilize existing technology. Biological means of incorporating modified carbohydrates into antibiotics has been achieved in the syntheses of novobiocin (14, 15), spinosyn (16), premithramycin (17), and oleandomycin (18). A rational genetic engineering approach requires a detailed knowledge of both the structural basis of substrate recognition and the biosynthetic reaction at each step of the pathway.

Chloroeremomycin (Fig. 1A), which is produced by the Actinomycete species Amycolatopsis orientalis, possesses the 4-epi isomer of vancosamine, L-epivanosamine (3-amino-2,3,6-trideoxy-3C-methyl-L-arabino-hexopyranose). It is attached to the glucose O-2' that is, in turn, attached to the -OH-Tyr⁶ of the glycopeptide. Sequencing of the A. orientalis gene cluster responsible for chloroeremomycin biosynthesis has identified open reading frames 14 and 23-26, encoding five enzymes that have been shown (19) to catalyze the biosynthesis of dTDP-L-epivancosamine from dTDP-6-deoxy-d-xylo-4-hexulose. The latter is the product of RmlB (dTDP-D-glucose 4,6-dehydratase) action in the dTDP-L-rhamnose biosynthesis pathway (20).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Carbohydrates and vancomycin. A, the structure of chloroeremomycin, a member of the vancomycin antibiotic family. The sugar 4-epivancosamine is highlighted in the two boxes. B, the pathway for the biosynthesis of dTDPL-epivancosamine from dTDP-glucose pro posed by Chen et al. (19). C, reaction scheme for the EvaD kinetic assay.

The structure of native EvaD to 1.5 Å and the structure of a dTMP EvaD complex to 1.4 Å have been determined and are presented in this study. They show that EvaD is indeed a member of the RmlC class of enzymes. However, EvaD is more than 200-fold less active in the 3',5' epimerization reaction than RmlC from Streptococcus suis. Examination of the active site of EvaD identified subtle changes in the position of a key Tyr residue, which may explain this altered activity. These results are in accord with the pathway shown by Chen et al. (19) and provide a structural basis for the redesign of a key step in dTDP-epivancosamine biosynthesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Enzyme Expression, Purification, Crystallization, and Data Collection—The expression, purification, and crystallization of EvaD have been described previously (23). The native x-ray data to 1.5 Å resolution were recorded at the European Synchrotron Radiation Facility (ESRF) BM14 Beamline at a wavelength of 0.933 Å using the ADSC Q4 CCD area detector. The beam size was 50 µm, and the crystal to detector distance was 130 mm. The data were recorded as 110 nonoverlapping 15-s 1 ° oscillations. The images were indexed and integrated in Mosflm, version 6.2 (24), as a primitive orthorhombic Laue group. Merging of the data, using DENZO SCALEPACK (25), and examination of systematic absences identified the crystal as belonging to space group P2₁2₁2 with cell dimensions a = 98.6, b = 72.0, c = 57.1, = = = 90°. A crystal of similar dimensions was soaked for 10 min in the cryoprotectant solution of 20% glycerol and mother liquor containing TDP-D-glucose. A full complex data set to 1.4 Å resolution was collected on this crystal at the ESRF (ID14.2). The data were recorded as 200 nonoverlapping 15-s 1° oscillations. This data set was integrated, merged, re-indexed, and scaled in the same space group as the native data set.

Structure Determination and Refinement—Starting phases for the native data were calculated by molecular replacement using RmlC from Salmonella enterica serovar Typhimurium as a search model (Protein Data Bank entry 1DZR [PDB] ). The program Molrep (26), as implemented in the CCP4 Program Suite (27), was used for molecular replacement using all the data in the resolution range 15-3 Å. For later cross-validation, 5% of the data were excluded prior to all refinement steps using the CCP4 program Uniquify.

The initial phases from molecular replacement were used for auto-tracing by the program ARP/wARP (28) using 100 cycles of building with 10 refinement cycles in between. The first building cycle identified 325 of the 404 residues with a connectivity index of 0.92; the procedure converged after 41 cycles identifying 389 residues in 8 separate chains and a connectivity index of 0.96. Electron density maps (F_o - F_c and 2F_o - F_c) were calculated and displayed in the graphics program O, version 7.0 (29), and the remaining 15 residues were fitted into the density manually.

Initial structure refinement was carried out in Refmac, version 5.0 (30), using the rigid body refinement option. Thermal liberation screw refinement, the addition of water and glycerol molecules to the structure, and finally anisotropic B factor refinement decreased the R_factor and R_free to the final values 14.4 and 17.8%, respectively. The complex structure was refined against the noncomplexed structure introducing the R_free flag from the apo data; the pyranose ring was not located in the experimental map. Refinement was carried out in Refmac, version 5.0, checking the maps and placing water molecules, glycerol, and finally dTMP in their respective places. The final R_factor and R_free values of the dTMP EvaD complex are 13.1 and 16.6%, respectively. Table I summarizes the data collection and structure refinement statistics. The coordinates of the structures as well as the structure factors have been deposited in the Protein Data Bank; the accession codes are 1OFN [PDB] (native) and 1OI6 [PDB] (dTMP complex).

The mutants were confirmed by DNA sequencing. The proteins were expressed and purified in the same way as the native protein (23), their integrity was confirmed by electrospray mass spectrometry, and correct folding was validated by CD spectroscopy.

Kinetic Assays—A three-enzyme coupled assay was carried out as described previously for the rhamnose pathway (22, 31). Dual epimerization by EvaD was measured by substituting EvaD for RmlC in the coupled assay. This assay contained RmlB and RmlD in excess and rate-limiting amounts of RmlC or EvaD. Enzyme activity was determined by measuring the oxidation of NADPH at A₃₄₀ by RmlD. This enzyme only recognizes the substrate after C-3' and C-5' epimerization, meaning that a measurable reaction rate shows that EvaD is capable of catalyzing the double epimerization. Fig. 1C gives an overview of the reaction scheme for the kinetic assay.

The three-enzyme assay contained 20 mM Tris, pH 7.5, 9 mM MgCl₂, 0.3 mM NADPH, 1.3 µM RmlB, 1 µM RmlD, and 0.03 µM RmlC. When EvaD was substituted for RmlC, significantly more enzyme had to be used to detect activity. The apparent K_m and k_cat of EvaD were determined using 12.5 µM EvaD. It was confirmed that EvaD, and not RmlB or RmlD, was rate-limiting for the EvaD assays. Protein concentration was determined by BCA assays, and purity was judged by SDS-PAGE. All of the assays were carried out in triplicate at 21 °C and repeated on several occasions. The background rate was established by measuring the NADPH oxidation of the enzyme assay mixture in the absence of dTDP-glucose and of the assay mixture containing dTDP-glucose but no RmlC/EvaD. Apparent K_m and k_cat were determined by varying the concentration of the initial substrate for the assay, dTDP-glucose, in the range of x (apparent K_m) to 10x (apparent K_m).

Deuterium Incorporation Studies—These experiments were performed and analyzed as described previously (21). In outline, following incubation with enzyme in D₂O, the keto-sugar nucleotide was reduced with sodium borohydride and cleaved with trifluoroacetic acid. The resulting reducing hexoses were further reduced with sodium borohydride to the corresponding alditols, per-O-acetylated, and analyzed by gas chromatography-mass spectrometry against an internal myo-inositol standard.

CD Spectroscopy—The mutants were analyzed by CD spectroscopy, and their spectra were compared with that of wild-type EvaD. The CD scans were carried out in the Protein Characterization Facility, Institute of Biomedical & Life Sciences, University of Glasgow. The spectra were recorded on samples at 1 mg/ml protein concentration in 20 mM Tris-HCl, pH 7.5. All of the mutants have spectra identical to that of the wild-type protein, indicating correct protein folding.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overall Structure of EvaD—The apo EvaD structure has been determined to 1.5 Å (Table I). The protein monomer is composed mainly of -sheets and has a jelly roll-like topology with overall dimensions: 47 x 34 x 51 Å. The topology of the monomers is essentially identical to that of authentic RmlC structures (32, 33), and the root mean square distances between the main chain of EvaD and those of published structures of RmlC fall within the range observed for authentic RmlC structures. Fig. 2 presents a sequence and structural alignment of the carbon -backbone of EvaD and selected RmlC homologues. The monomer can be divided into three separate regions: the N-terminal, core, and C-terminal regions. The N terminus (residues 1-47) consists of an antiparallel -sheet (1-3) and a two-turn -helix. The core of the monomer consists of two twisted antiparallel -sheets (5-13), which form a flattened barrel. One end of the barrel is open, and the entrance is lined with polar residues; the other side is obscured by -strands that fold over the entrance. A number of hydrophobic residues in this part of the polypeptide chain seal the entrance to the barrel. In addition, the middle of the barrel is packed with a cluster of hydrophobic residues. The C-terminal region consists of residues 168-202 and comprises two helical turns, three short -strands, and a short -helix. As with RmlC, EvaD is a homodimer with an interface formed by the antiparallel interaction of two -strands from differing monomers (Fig. 3A). The dimer interface buries 17% of the accessible surface area of the monomer, calculated using the Protein-Protein Interactions Server at University College, London by Laskowski et al. (www.biochem.ucl.ac.uk/bsm/PP/server) (34-36).

View larger version (70K): [in this window] [in a new window]   FIG. 2. Sequence and structural alignment of EvaD and several RmlC proteins from different species. The conserved residues are boxed. Residues marked in white on black backgrounds are absolutely conserved throughout all species. Bold letters indicate conserved or conservatively substituted residues. denotes the presence of -helices, denotes that of -sheets, and residues in turns are denoted with T.

View larger version (30K): [in this window] [in a new window]   FIG. 3. The structure of EvaD. A, secondary structure of the EvaD dimer with dTMP bound in the nucleotide pocket. The chains are colored green to red and cyan to green, for N to C terminus. B, the Fo - Fc electron density map at a level of 1 shows clearly that dTMP is bound to the active site of EvaD. This figure shows the residues involved in nucleotide binding, Arg23 and Leu26 are part of one monomer and Arg60, Arg169, and Tyr139 of the other. C, a cross-eyed stereo diagram of the active site of S. suis superimposed upon the active site of EvaD. dTDP-xylose from the S. suis structure is shown for reference (32). Carbon is green in RmlC from S. suis and blue in EvaD. The catalytic Tyr residue is locked into different conformation in RmlC and EvaD. Tyr140 in RmlC adopts the g+ conformation and is blocked by Tyr138 (Met131 in EvaD) from adopting the g- conformation. Tyr133 in EvaD adopts the g- conformation and is blocked by Leu135 (Val142 in RmlC).

No gross structural changes in EvaD between the apo and dTMP complex structures can be observed. The root mean square distance between the main chains is 0.2 Å, and the root mean square deviation in B factors is 2 Ų. The thymidine ring is sandwiched between Tyr¹³⁹ and Leu^26*, where the asterisk denotes that this particular residue is part of the other monomer. This differentiates EvaD from the RmlC enzymes in which two aromatic residues, one from each monomer, sandwich the thymidine. Consequently the plane of the thymidine ring is offset by 50° relative to the plane of thymidine ring in the RmlC complexes (32). The altered nucleotide orientation perturbs the positions of the ribose ring and phosphate groups. Despite these changes, it is clear that the dTMP portion of the ligand is bound in a very similar manner to that seen in RmlC enzymes (32). The ribose ring is not specifically recognized. The phosphate group of dTMP is bound by Arg¹⁶⁹ and Arg^23*. The absolutely conserved Arg⁶⁰ is ideally positioned to bind the second phosphate group present in the substrate.

Catalytic Site—As yet, we have been unable to obtain a complex with a pyranose ring detectable at the active site of EvaD. Therefore we have used the dTMP complex of EvaD and the structures of S. suis RmlC with substrate analogues (dTDP-glucose and dTDP-xylose) to generate models of pyranose molecules in the EvaD catalytic site. This is a valid approach as the majority of the substrate atoms overlap, and all of the dTMP atoms align exactly when the protein molecules are overlapped. The dTDP-glucose and dTDP-xylose RmlC complex structures are broadly similar (32), but there are subtle differences between the contacts of the two sugars resulting from a rotation of the pyranose ring in the active site. Therefore, EvaD models based on the structures of both dTDP-glucose and dTDP-xylose RmlC complexes were examined. The sugar nucleotide is bound in a U-shaped conformation (32). The EvaD catalytic site is slightly larger than that of RmlC enzymes because the loss of a thymidine stacking aromatic residue opens up the binding site. In the catalytic site the modeled pyranose ring sits above the ND1 atom of His⁶³; thus the axial groups at the C-3' and C-5' positions of the sugar point toward this residue, which is ideally positioned to function as the catalytic base. In the dTDP-xylose complex the O-4' atom of the pyranose, where the enolate forms, is close to residues His¹²⁰ and Lys⁷³, which have already been proposed to stabilize the reaction intermediate (32). His¹²⁰ is not an absolutely conserved residue (found as Asn) and was therefore eliminated as either a base or an acid (32). The methyl group at C-6' points toward a hydrophobic region made up of Phe¹²², Met¹³¹, and the aliphatic portion of the side chain of Arg⁶⁰. At first glance there is no difference between the interactions and orientation of the pyranose ring in EvaD and that of our previously reported RmlC complexes (32). However, closer inspection of EvaD revealed a marked difference: the OH group of the catalytic Tyr¹³³ clashes with the C-5' methyl group (<3 Å) and any equatorial substituent (<2 Å) on either glucose or xylose. The reason for this change is that the Tyr residue in EvaD has a different orientation to that in any of the RmlC structures (Fig. 3C).

Biochemical Characterization of EvaD in the RmlC Reaction—The apparent k_cat for the double epimerization of dTDP-6-deoxy-d-xylo-4-hexulose catalyzed by EvaD is more than 2 orders of magnitude lower than that observed for RmlC from either S. suis or S. enterica serovar Typhimurium (Table II). Mutating residues His⁶³ and Tyr¹³³ to Ala and Phe, respectively, abolishes detectable epimerization activity. This finding is consistent with their roles as the catalytic base and acid, previously identified by studies on RmlC enzymes (32). Their inactivity provides a control for the assay and allows us to identify that EvaD possesses real but low double epimerization activity. This is surprising because the RmlC from S. enterica serovar Typhimurium is more similar in sequence and structure to EvaD than it is to the RmlC enzyme from S. suis. Yet the RmlC enzymes from S. enterica serovar Typhimurium and S. suis have very similar kinetic parameters (32), whereas EvaD is quite different. The EvaD mutant M131F shows a 1.5-fold increase in apparent k_cat despite its 1.6-fold decrease in apparent substrate affinity.

View larger version (32K): [in this window] [in a new window]   FIG. 4. Deuterium incorporation. A, mass spectrometry fragments of alditol per-acetates associated with deuterium incorporation. Starting material: 217/231; selective D incorporation at C-5': 217/232; selective D incorporation at C-3': 218/232; double D incorporation at C-3' and C-5': 218/233. B, enzymatic incorporation of deuterium into dTDP-6-deoxy-d-xylo-4-hexulose. The diagonally hatched bars represent no deuterium incorporation, horizontally hatched bars represent C-3' incorporation, vertically hatched bars represent deuterium incorporation at C-5', and solid black bars represent double incorporation at C-3' and C-5' positions. The control contains no enzyme, native RmlC is from S. suis, and other proteins are EvaD. The error bars represent the standard deviation between measurements.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The structure of EvaD shows that although Tyr¹³³ is conserved in the protein sequence, it is not structurally conserved (Fig. 3C). In all RmlC structures the positioning of this residue is identical; the Tyr residue is found in the less energetically favorable and less common g+ conformation with ₁ and ₂ torsion angles of 107° and 60°, respectively (38). The catalytic Tyr¹³³ of EvaD displays torsion angles in agreement with the more commonly observed and energetically more favorable g- rotamer (₁ = 53° and ₂ = 117°). In RmlC enzymes the Tyr cannot adopt the g- conformation because an aromatic residue (Tyr¹³⁸ in S. suis) occupies the space. Examination of the sequences of more than 100 RmlC homologues determined that an aromatic residue is found in 71%, a Val is found in 26%, and an Ile is found in 3% of the enzymes at this position. All three residues have a tertiary C atom and would sterically interfere with a g- conformation of the catalytic Tyr residue. In EvaD, the blocking aromatic residue is changed to Met¹³¹ whose secondary C atom is positioned in such a way as to create a pocket for the g- conformer. Thus, in RmlC and EvaD the position of the catalytic Tyr appears to be locked into different conformations. The Tyr¹³³ phenol group in EvaD, from which the proton is donated, is more than 4.5 Å away from its position in RmlC. Thus, EvaD cannot bind the RmlC substrate dTDP-6-deoxy-d-xylo-4-hexulose in an identical manner to RmlC. In EvaD, the RmlC substrate will have to shift in the active site to avoid clashing with Tyr¹³³ and to be in position to receive the proton from Tyr¹³³. To test our hypotheses about Tyr¹³³, we made two mutants (data in Table I). The Y133F mutant is effectively inactive, confirming that this residue is of key importance in EvaD. To reposition Tyr¹³³ within the active site, we constructed a M131F mutant. The structure of the native enzyme predicts that this mutation will block the pocket that accommodates the g- conformation of Tyr¹³³ seen in EvaD, presumably forcing the Tyr to adopt the g+ conformation. The mutated protein shows an improvement in turnover in the RmlC reaction and a significant increase in deuterium incorporation at the C-3' position. We regard these results as evidence that fine tuning of the Tyr¹³³ position allows EvaD to distinguish between the RmlC substrate and its correct substrate dTDP-3-amino-2,3,6-trideoxy-3C-methyl-D-erythro-hexopyranosyl-4-ulose. This discrimination may be required because it is not in the interest of the organism to epimerize the true RmlC substrate to any significant degree because dTDP-6-deoxy-d-xylo-4-hexulose is a substrate for the dTDP-L-epivancosamine pathway, and its double epimerization would reduce the yield of L-epivancosamine by creating an alternative biosynthetic route. The subtle change in the position of an active residue is an elegant method of achieving such discrimination.

	FOOTNOTES

 
The atomic coordinates and structure factors (codes 1OFN [PDB] and 1OI6 [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a Wellcome Trust program grant (to J. H. N.). 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.

^|| Present address: Dept. of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0358.

^** A Biotechnology Biological Sciences Research Council Career Development Fellow. To whom correspondence should be addressed. Tel.: 44-1334-463792; Fax: 44-1334-462595; E-mail: naismith{at}st-and.ac.uk.

	ACKNOWLEDGMENTS

 
We thank the reviewer and editor for constructive suggestions and critical comments, which considerably improved the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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