J Biol Chem, Vol. 274, Issue 35, 25069-25077, August 27, 1999

Characterization of dTDP-4-dehydrorhamnose 3,5-Epimerase and dTDP-4-dehydrorhamnose Reductase, Required for dTDP-L-rhamnose Biosynthesis in Salmonella enterica Serovar Typhimurium LT2^*

Michael Graninger, Bernd Nidetzky^§, David E. Heinrichs^¶, Chris Whitfield^¶, and Paul Messner^¶**

From the  Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria, the ^§ Institut für Lebensmitteltechnologie, Universität für Bodenkultur Wien, A-1190 Wien, Austria, and the ^¶ Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ontario, N1G 2W1 Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The thymidine diphosphate-L-rhamnose biosynthesis pathway is required for assembly of surface glycoconjugates in a growing list of bacterial pathogens, making this pathway a potential therapeutic target. However, the terminal reactions have not been characterized. To complete assignment of the reactions, the four enzymes (RmlABCD) that constitute the pathway in Salmonella enterica serovar Typhimurium LT2 were overexpressed. The purified RmlC and D enzymes together catalyze the terminal two steps involving NAD(P)H-dependent formation of dTDP-L-rhamnose from dTDP-6-deoxy-D-xylo-4-hexulose. RmlC was assigned as the thymidine diphosphate-4-dehydrorhamnose 3,5-epimerase by showing its activity to be NAD(P)H-independent. Spectrofluorometric and radiolabeling experiments were used to demonstrate the ability of RmlC to catalyze the formation of dTDP-6-deoxy-L-lyxo-4-hexulose from dTDP-6-deoxy-D-xylo-4-hexulose. Under reaction conditions, RmlC converted approximately 3% of its substrate to product. RmlD was unequivocally identified as the thymidine diphosphate-4-dehydrorhamnose reductase. The reductase property of RmlD was shown by equilibrium analysis and its ability to enable efficient biosynthesis of dTDP-L-rhamnose, even in the presence of low amounts of dTDP-6-deoxy-L-lyxo-4-hexulose. Comparison of 23 known and predicted RmlD sequences identified several conserved amino acid residues, especially the serine-tyrosine-lysine catalytic triad, characteristic for members of the reductase/epimerase/dehydrogenase protein superfamily. In conclusion, RmlD is a novel member of this protein superfamily.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial cell-surface glycoconjugates are essential for survival of pathogenic bacteria and interactions between bacteria and host. Consequently, there is reason to believe that inhibitors directed against target reactions in surface glycoconjugate assembly may provide viable alternate therapeutic approaches. However, bacterial cell surface glycoconjugates show remarkable structural diversity due to variations of the sugar components, linkages, and substitutions. A successful strategy requires identification of enzymes and pathways unique to bacteria, yet present within a wide spectrum of bacterial species. One such target is the synthesis of the activated form of L-rhamnose, dTDP¹-L-rhamnose. L-Rhamnose is found in polysaccharides from strains of important human pathogens such as Salmonella, Shigella, Burkholderia, and streptococci, as well as in plant-associated bacteria including Xanthomonas and Rhizobium. The primary structures of many of these glycoconjugates have been reported (see the Complex Carbohydrate Structure Data base). L-Rhamnose is also found in many surface layer glycoproteins from Bacillaceae (1), in the linkage unit that joins the mycolylarabinogalactan complex to peptidoglycan in mycobacteria (2) and in some mycobacterial glycopeptidolipids (3). Examination of gene data bases also indicates the presence of the structural genes for enzymes involved in dTDP-L-rhamnose synthesis in strains where the rhamnose-containing structure is not necessarily resolved, for example, in Enterococcus faecalis (4), Leptospira interrogans serovar Copenhageni (5), and some members of the archaea.

The pathway for the biosynthesis of dTDP-L-rhamnose from glucose 1-phosphate and thymidine triphosphate was proposed in the early 1960's by Glaser and Kornfeld (6, 7) although the enzymes were not specifically identified. Genetic data indicates that the pathway requires four genes, rmlABCD with the prototypes being identified in the lipopolysaccharide O-antigen biosynthesis (rfb) gene cluster from Salmonella enterica serovar Typhimurium LT2 (8).

The reaction steps and the genes required for dTDP-L-rhamnose biosynthesis (9, 10) were: 1) dTTP + D-Glc-1-P  dTDP-D-Glc + PP_i (glucose-1-phosphate thymidyltransferase, RmlA: EC 2.7.7.24); 2) dTDP-D-Glc  dTDP-6-deoxy-D-xylo-4-hexulose (dTDP-D-glucose 4,6-dehydratase, RmlB: EC 4.2.1.46); 3) dTDP-6-deoxy-D-xylo-4-hexulose  dTDP-6-deoxy-L-lyxo-4-hexulose (dTDP-4-dehydrorhamnose 3,5-epimerase, RmlC: EC 5.1.3.13); 4) dTDP-6-deoxy-L-lyxo-4-hexulose + NAD(P)H  dTDP-L-rhamnose + NAD(P)⁺ (dTDP-4-dehydrorhamnose reductase, RmlD: EC 1.1.1.133).

Recently, the first two enzymes in the pathway, glucose-1-phosphate thymidyltransferase (RmlA) (11) and dTDP-D-glucose 4,6-dehydratase (RmlB) (12) were analyzed in detail. Overexpressed gene products were used for enzymatic synthesis of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose with varying yield (12, 13). Although RmlC and D are required for the conversion of dTDP-6-deoxy-D-xylo-4-hexulose to dTDP-L-rhamnose, the definitive assignment of individual activities has not been done, and the mechanism has not been elucidated. This is a clear limitation for studies where inhibitor development is the ultimate goal.

In this report we describe the purification, characterization, and unequivocal assignment of dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC) and dTDP-4-dehydrorhamnose reductase (RmlD) using the overexpressed enzymes from S. enterica serovar Typhimurium LT2. The following reaction scheme for the conversion of dTDP-6-deoxy-D-xylo-4-hexulose to dTDP-L-rhamnose by RmlC and D is proposed (Reaction 1).

View larger version (8K): [in this window] [in a new window]   Reaction 1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Thymidine monophosphate (dTMP), thymidine diphosphate (dTDP), thymidine triphosphate (dTTP), dTDP-D-glucose, CDP-D-glucose, UDP-D-glucose, ADP-D-glucose, GDP-D-glucose, NADH, NADP⁺, NAD⁺, dithiothreitol, glucose 1-phosphate, and Cibacron blue-Sepharose CL-4B were obtained from Sigma (Sigma-Aldrich-Fluka GmbH, Vienna, Austria). NADPH was from Biomol (Hamburg, Germany). Mono Q HR 5/5, phenyl-Superose HR 5/5, DEAE-Sephacel, and Sephadex G-10 were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Bio-Gel HT was from Bio-Rad (Vienna, Austria).

Analytical Techniques

Nucleotide-activated sugars were analyzed on a CarboPac PA-1 column by the method of Köplin et al. (14). Monosaccharides were analyzed on a CarboPac PA-1 column as described previously (15). Determination of the molecular weight of native proteins was done on a UltroPac TSK G3000SW column (LKB, 7.5 × 300 mm) in 0.2 M ammonium acetate, pH 7.0. Bovine serum albumin (67,000), ovalbumin (43,000), chymotrypsinogen A (25,000), and RNase A (13,700) were used as reference proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using slight modifications (16) of the original method of Laemmli (17). Nondenaturing PAGE was performed in 50 mM Tris/citrate buffer at pH 7.0. The resolving gels contained 12.5%, and the stacking gels 5% polyacrylamide (3% cross-linking). Ammonium persulfate and N,N,N',N'-tetramethylethylenediamine were removed by prerunning gels at 200 V for 1 h with two changes of Tris/citrate buffer. Electrophoresis was performed using a constant voltage (200 V) at 10 °C for 4 h. Gels were silver-stained using a method described elsewhere (18). Protein concentration was determined by the method of Bradford (19). Fluorescence spectroscopy was performed at 25 °C in a Hitachi F-2000 spectrofluorometer and at excitation and emission wavelengths as indicated. Radioactivity counting was done in Ultima Gold liquid scintillation mixture (Packard Instrument Co., Meriden, CT) on a Packard Tri-Carb 1900 CA liquid scintillation counter.

Bacterial Strains and Growth Conditions

Plasmids were routinely maintained in Escherichia coli DH5 (K-12 F 80d lacZM15 endA1 recA1 hsdR17 (r_Km_K) supE44 thi-1 gyrA96 relA1 (lacZYA-argF) U169) (20). For enzyme overexpression, E. coli BL21 (DE3) (F ompT hsdS_B (r_Bm_B) gal dcm (DE3); Novagen, Madison, WI) was used. All strains were routinely grown in Luria-Bertani (LB) medium. Media were supplemented, where required, with kanamycin (K_m; 30 µg/ml). Cultures were grown at 37 °C with or without agitation.

Construction of Plasmids

DNA sequence for the complete rfb gene cluster from S. enterica strain LT2 was obtained from the GenBank (Ref. 8; accession number X56793). The order of the dTDP-L-rhamnose biosynthesis genes is rmlBDAC. The four open reading frames were individually amplified by polymerase chain reaction using primers designed to introduce a unique NcoI or NdeI site (underlined below) overlapping the initiating ATG codon. A downstream SstI site was introduced to facilitate cloning of the amplified fragment. Primers were synthesized with the following sequences at the Guelph Molecular Supercentre: RMLB1(forward), 5'-TGGAATAGAAccaTGgAGATACTT-3' (NcoI); RMLB2 (reverse), 5'-TTACTgAgcTCACCGCAAAACTCTT-3' (SstI); RMLD1 (forward), 5'-GAAGGACGCCAcatATGAATATCTT-3' (NdeI); RMLD2 (reverse), 5'-TTgAgcTCCATTTCTCATTCATAGA-3' (SstI); RMLA1 (forward), 5'-AGAAATGcAtATGAAAACGCGTAAG-3' (NdeI); RMLA2 (reverse), 5'-TTTgagCTCTAAGATCAAGACATCT-3' (SstI); RMLC1 (forward), 5'-AGGTTTAcAtaTGATGATTGTGATT-3' (NdeI); RMLC2 (reverse), 5'-TTCgagCTCTCTACCGGAAAATTCA-3' (SstI). Lowercase letters indicate nucleotides introduced to construct appropriate restriction sites (in parentheses); the restriction sites are underlined.

Polymerase chain reaction amplifications were performed using a GeneAmp polymerase chain reaction system 2400 (Perkin-Elmer, Norwalk, CT). Chromosomal DNA from S. enterica LT2 (1 µg/µl) was obtained from Dr. Wendy J. Keenleyside. For the amplification reactions, 2.5 units of PwoI DNA polymerase (Roche Molecular Biochemicals), 0.5 µg of template DNA, 200 µmol of each deoxynucleotide triphosphate, and 25 µmol of the corresponding synthetic nucleotide primers were used. Twenty cycles were used. Optimal MgSO₄ concentration and reaction temperatures and times were individually determined for each polymerase chain reaction product. The product sizes for rmlA (943 base pairs), rmlB (1, 222 base pairs), rmlC (627 base pairs), and rmlD (966 base pairs) were those predicted from their respective nucleotide sequences.

The amplified fragments were digested with the appropriate restriction endonucleases, according to the manufacturer's recommendations and cloned in plasmids pET-28a(+) (rmlB) or pET-30a(+) (rmlA,C,D) (Novagen, Madison, WI). The procedures for manipulation of DNA and for ligation were those described by Sambrook et al. (20). Electrotransformations of E. coli DH5 and BL21 (DE3) were performed using published protocols (21) with a Gene Pulser apparatus (Bio-Rad). Transformants were selected on LB/K_m plates. Plasmids were column-purified using QIAGEN spin columns (QIAGEN, Chatsworth, CA) according to the manufacturers instructions. The sequence of each cloned rml gene was determined and confirmed to be identical to the authentic chromosomal copy. Plasmid DNA sequencing was performed by automated sequencing at the Guelph Molecular Supercentre.

Sequence Analyses

Nucleotide and protein sequences were analyzed using online analysis tools, including BLAST (Basic local alignment search tool; Ref. 22) and Clustal W 1.6 (Refs. 23 and 24) using default parameter settings. For protein family analysis and motif analysis, the Prosite data base (25) was used.

Expression of Genes in the rml Operon

The pET vector-based constructs place the cloned rml genes under the control of a T7-promoter. For overexpression of the rml gene products, an isopropyl-1-thio--D-galactopyranoside-inducible T7 RNA polymerase is supplied by the host, E. coli BL21 (DE3). Isopropyl-1-thio--D-galactopyranoside induction of E. coli BL21 (DE3) transformed with pET constructs carrying each rml gene, led to expression of RmlA, RmlB, RmlC, and RmlD as the predominant proteins in the induced cultures. For large-scale enzyme preparations, up to 500 ml of LB/K_m medium was inoculated with 1% of an overnight culture and cultivated at 37 °C on a rotary shaker. After 3 h, isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM and incubation was continued for 3 h. The bacterial cells were then collected by centrifugation at 7,000 rpm for 6 min at 4 °C. The cell pellets were washed twice with cold 10 mM Tris-HCl buffer, pH 8.0. Before sonication, the buffer was adjusted by addition of dithiothreitol and MgCl₂ to final concentrations of 1 and 10 mM, respectively. Sonication on ice was carried out 6 times for 10 s with intervals of 20 s. Large debris were removed by centrifugation at 5,000 rpm for 10 min at 4 °C. The cell-free supernatants were carefully removed and centrifuged at 60,000 rpm for 40 min at 4 °C in a Ti 70.1 rotor in a Beckman LE-80 ultracentrifuge. Supernatants were stored at 20 °C following addition of glycerol to a final concentration of 44%.

(Eq. 3)

Enzyme Assays

Glucose-1-phosphate thymidyltransferase (EC 2.7.7.24, RmlA) and dTDP-D-glucose 4,6-dehydratase (EC 4.2.1.46, RmlB) activities were assayed as described by Kornfeld and Glaser (6) and Vara and Hutchinson (26), respectively.

The specific assay for determination of RmlC typically contained 45 mM potassium phosphate buffer, pH 7.0, 9 mM MgCl₂, 0.18 mM dTDP-6-deoxy-D-xylo-4-hexulose, 0.072 mM NAD(P)H, a 20-fold molar excess of RmlD for determination of RmlC, and an appropriate volume of RmlC in a total volume of 550 µl. Assays for RmlD were done using a similar reaction mixture but incorporating a 100-fold molar excess of RmlC. Time absorption plots were recorded at 25 °C in a Beckman DU65 spectrophotometer or a Hitachi U-2010 spectrophotometer. Enzyme activities were calculated from the linear decrease of the absorption at 340 nm. Assays with low amounts of NAD(P)H were analyzed in a Hitachi F-2000 spectrofluorometer with an excitation at 340 nm and recording emission at 460 nm. One unit of RmlC or RmlD activity refers to 1 µmol of NAD(P)H consumption per minute using these assay conditions. Controls without addition of dTDP-6-deoxy-D-xylo-4-hexulose, or enzyme, were analyzed by the same procedure.

Equilibrium Analysis

For determination of the thermodynamic equilibrium constants of the reaction described in reaction steps 3 and 4, the reverse reaction starting from dTDP-L-rhamnose was used. Assays contained 0.05 to 0.18 mM dTDP-L-rhamnose, 0.03 to 0.4 mM NAD⁺, 0.16 µM RmlD in 50 mM potassium phosphate buffer, pH 7.0, or 50 mM ethanolamine-HCl buffer, pH 9.0. The assays were performed with, and without, addition of RmlC (0.6 µM). Equilibrium constants were calculated as: K_eq,RmlD = [NAD⁺][dTDP-L-rhamnose]/[NADH][dTDP-6-deoxy-L-lyxo-4-hexulose][H⁺] and K_eq,RmlD × K_eq,RmlC = [NAD⁺][dTDP-L-rhamnose]/[NADH][dTDP-6-deoxy-D-xylo-4-hexulose][H⁺].

pH Optima

For determination of the pH dependence of enzyme activity, RmlC and RmlD were assayed in different buffers ranging from pH 5.5 to 11.0 (50 mM potassium phosphate buffer, pH 5.5-8.5, and 50 mM ethanolamine-HCl buffer, pH 8.5-11.0) without addition of MgCl₂. To determine the profile of enzyme stability versus pH, enzymes were diluted in various buffers (potassium phosphate buffer, pH 5.5-8.0) and incubated at 37 °C for 20 min. Residual activities were determined at pH 7.0 after exchange of the buffer.

Kinetic Analyses

Measurements of kinetic data were performed using the spectrophotometric and spectrofluorometric assays described above. The kinetic constants, K_m and k_cat, where K_m is the apparent Michaelis constant and k_cat is the turnover number, were obtained by fitting the experimental data to the equation (27),

(Eq. 1)

using the program Sigma Plot (Jandel, Erkrath, Germany). k_cat (1/s) was calculated from the maximum initial velocity v_max (µmol/(liter·s)) as,

(Eq. 2)

where E is the total enzyme concentration (µmol/liter). A molecular mass of 20.6 and 32.6 kDa was used for RmlC and RmlD, respectively. For RmlC, the initial velocities were recorded for the concentration range 0.036 to 0.36 mM dTDP-6-deoxy-D-xylo-4-hexulose. The RmlC concentration was 16.6 nM and RmlD was used in 20-fold molar excess. For RmlD, NADH and NADPH concentrations were 0.0015 to 0.109 mM and dTDP-6-deoxy-D-xylo-4-hexulose concentration was 0.18 mM. The RmlD concentration was 7.5 nM and RmlC was used in 100-fold molar excess. Activities at NAD(P)H concentrations below 0.01 mM were measured spectrofluorometrically.

For the analysis of the reaction mechanism of RmlD the concentrations of NADPH and dTDP-6-deoxy-D-xylo-4-hexulose were varied between 0.007 and 0.11 mM, and 0.091 and 0.36 mM, respectively. The experimental data were fit to,

for a sequential reaction mechanism (27, 28). For an ordered mechanism, k_I represents the dissociation constant of the first substrate. The correlation coefficients of nonlinear regression were usually 0.98 or better. Variance analysis and other statistics provided by the program showed that Equation 3 adequately fits the data.

Purification of dTDP-4-dehydrorhamnose 3,5-Epimerase

To prevent oxidative damage of proteins during the purification process, all buffers contained 0.5 mM dithiothreitol. Fractions were collected on ice.

Step 1: Hydroxyapatite Chromagraphy-- Cell-free lysates were applied to a Bio-Gel HT column (1.6 × 10 cm) at a flow rate of 1 ml/min. Proteins were eluted using the following gradient: 0-20 ml, 10 mM potassium phosphate buffer, pH 6.8; 20-100 ml, 0-100% 200 mM potassium phosphate buffer, pH 6.8. Two-ml fractions were collected. Fractions showing enzyme activity eluted at approximately 0.06 M potassium phosphate and these were combined and adjusted to 1 M ammonium sulfate.

Step 2: Hydrophobic Interaction Chromatography-- Pooled enzyme fractions from step 1 were applied to phenyl-Superose HR 5/5 column equilibrated with 50 mM potassium phosphate buffer, pH 7.0, containing 1 M ammonium sulfate. Proteins were eluted at a flow rate of 0.5 ml/min using the following gradient: 0-10 ml, 1 M ammonium sulfate; 10-30 ml: 1-0 M ammonium sulfate. One-ml fractions were analyzed for enzyme activity. Enzymatically active fractions eluted at 0.2 M ammonium sulfate and these were pooled and dialyzed overnight at 4 °C against several exchanges of 20 mM Tris-HCl buffer, pH 7.7.

Step 3: Ion Exchange Chromatography-- The enzyme preparation from step 2 was applied to a Mono Q HR 5/5 column equilibrated with 20 mM Tris-HCl buffer, pH 7.7. Proteins were eluted at a flow rate of 1 ml/min by the following gradient: 0-10 ml, 20 mM Tris-HCl buffer, pH 7.7; 10-30 ml, 0 to 0.5 M KCl. Fractions showing enzyme activity eluted at 0.28 M KCl. These were combined, adjusted to 44% glycerol, and stored at 20 °C until use. The purified RmlC enzyme preparation (86% yield; approximately 12.5 mg/liter culture) showed a specific activity of approximately 21 units/mg.

Purification of dTDP-4-dehydrorhamnose Reductase

Step 1: Cibacron Blue-Sepharose Chromatography-- Cell-free LysatesCell-free lysates (stored without addition of glycerol) were diluted 1:1 in 50 mM Tris-HCl buffer, pH 7.7, and up to 10-ml aliquots were applied to a Cibacron blue-Sepharose CL-4B column (1.5 × 5 cm) at a flow rate of 2.5 ml/min. The column was washed with 50% ethylene glycol in 50 mM Tris-HCl buffer, pH 7.7, and 0.3 M KCl in the same buffer. Most of the proteins in the lysate did not bind to the matrix, while RmlD was absorbed and subsequently eluted in 1.5 mM KCl. The active fractions were dialyzed overnight as described.

Step 2: Ion Exchange Chromatography-- This was performed as described above. RmlD eluted at 0.14 M KCl. The purified RmlD enzyme preparation (70% yield; approximately 20 mg/liter culture) showed a specific activity of approximately 44 units/mg.

Synthesis of Nucleotide Activated Monosaccharides

Reaction mixtures for synthesis of dTDP-6-deoxy-D-xylo-4-hexulose (12, 13) contained approximately 20 µmol of dTDP-D-glucose and 2 units of RmlB in 1 ml of 20 mM Tris-HCl buffer, pH 7.7. Incubation was performed at 25 °C for 1 h. The reaction was stopped by addition of 1 ml of absolute ethanol and precipitated proteins were removed by centrifugation. The product was desalted on a Sephadex G-10 column (1.5 × 120 cm), lyophilized, and stored at 20 °C until use. UV spectroscopy at 320 nm performed in 0.1 M NaOH was used to determine concentration of dTDP-6-deoxy-D-xylo-4-hexulose. Quantitation is based on the characteristic absorption of the 4-keto group. Following reduction with NaBH₄ and hydrolysis, the monosaccharides obtained from dTDP-6-deoxy-D-xylo-4-hexulose were analyzed by high performance anion exchange chromatography with pulsed electrochemical detection (HPAEC/PED) (15).

For synthesis of dTDP-6-deoxy-L-lyxo-4-hexulose, 1 µmol dTDP-D-glucose was incubated at 25 °C for 30 min with 1 unit each of RmlB and RmlC in 500 µl of 30 mM Tris-HCl buffer, pH 7.0, at 25 °C for 30 min. The enzymes were removed by ultrafiltration and the reaction mixture containing both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-6-deoxy-L-lyxo-4-hexulose was desalted as described above. The amount of dTDP-6-deoxy-L-lyxo-4-hexulose in the resulting preparation was determined by conversion to dTDP-L-rhamnose in the presence of RmlD and spectrofluorometric measurement of the decrease of NADH. Direct proof for the presence of free dTDP-6-deoxy-L-lyxo-4-hexulose was obtained by reducing the 4-keto group with NaB[³H]₄ (100 mCi/mmol, American Radiolabeled Chemicals, St. Louis, MO). Following hydrolysis and HPAEC/PED of the resulting monosaccharides, fractions (0.25 ml) were analyzed for radioactivity.

dTDP-L-rhamnose was synthesized from dTDP-D-glucose using RmlB, RmlC, and RmlD in the presence of low amounts of NAD⁺. NADH was regenerated using NAD⁺-dependent formate dehydrogenase from Candida boidinii (ASA Spezialenzyme GmbH, Braunschweig, Germany). A typical reaction mixture contained 20 µmol of dTDP-D-glucose, 1 µmol of NAD⁺, 100 µmol of ammonium formate, 1 unit of each RmlB, RmlC, and RmlD, and 3.5 units of formate dehydrogenase in a total volume of 5 ml of 0.1 M Tris-HCl buffer, pH 7.0. After reaction at 25 °C for 2 h, proteins were removed by ultrafiltration in an Amicon model 8050 cell using a Millipore PLGC 10-kDa ultrafiltration membrane. dTDP-L-rhamnose was purified by anion exchange chromatography on a DEAE-Sephacel column as described previously (15) and desalted as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of the Cloned Protein Products in E. coli-- Isopropyl--D-thio-galactopyranoside-induction of E. coli BL21 (DE3) cells, transformed with pET-derivatives carrying each of the rmlABCD genes, led to expression of RmlA, RmlB, RmlC, and RmlD as the predominant proteins in the induced cultures (Fig. 1). Limited amounts of these proteins were also present in the noninduced cultures (not shown). The majority of each Rml protein was found in the cell-free supernatant fraction. This, together with their activities (see below), indicates that these proteins are located in the cytoplasm. The residual amounts of RmlC and RmlD were sedimented during removal of cellular debris from the ultrasonicated cell lysate. Electron microscopy of ultrathin-sectioned intact cells confirmed formation of inclusion bodies (not shown), presumably containing the enzyme confined to the particulate fraction. Since sufficient amounts of soluble protein was obtained, inclusion bodies were not pursued further for protein purification.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE analysis showing overexpression and purification of enzymes involved in dTDP-L-rhamnose biosynthesis. Cell-free supernatants from vector control (E. coli BL21 (DE3) transformed with pET-28a(+) (lane 2), and induced cells overexpressing RmlA (lane 3), RmlB (lane 4), RmlC (lane 5), and RmlD (lane 7). Purified enzyme preparations after Mono Q chromatography of RmlC (lane 6) and RmlD (lane 8). Molecular weight markers (lane 1): bovine serum albumin (66,300), glutamate dehydrogenase (55,400), lactate dehydrogenase (36,500), carbonic anhydrase (31,000), trypsin inhibitor (21,500), and lysozyme (14,400). Each lane corresponds to approximately 2.5 µg of total protein.

The expressed Rml enzymes showed in SDS-PAGE apparent molecular masses of 31.8, 42.9, 23.4, and 34.8 kDa, respectively. These values correspond closely to the predicted translation products based upon available nucleotide sequence data (32.5, 40.7, 20.6, and 32.6 kDa, respectively) (8). By gel permeation chromatography on an UltroPac TSK G3000SW column, the native molecular mass of RmlC was determined to be 40.6 kDa. This is consistent with RmlC forming a dimer (calculated molecular mass = 41.2 kDa). The molecular mass of RmlD, determined on a silica column was 41.5 kDa, a value inconsistent with the calculated molecular mass for either the monomer (32.6 kDa), or the dimer (65.2 kDa). The aberrant molecular mass estimated for RmlD may result from interactions between RmlD and the chromatographic resin. For example, the hydrophobic protein chymotrypsinogen A has been shown to bind tightly to silica matrices resulting in retarded elution and accordingly a lower apparent molecular mass (29). Comparison of chymotrypsinogen A and RmlD by analytical hydrophobic interaction chromatography on a phenyl-Superose HR 5/5 column revealed that RmlD elutes at even lower (NH₄)₂SO₄ concentrations than chymotrypsinogen A (0.5 and 0.1 M, respectively), indicating higher hydrophobicity of RmlD. In conclusion, the apparent molecular mass of RmlD, as determined by size exclusion chromatography to be 41.5 kDa, may correspond to at least a dimer.

Activity of the Overexpressed Rml Enzymes-- The correct folding and functionality of the overexpressed dTDP-L-rhamnose biosynthesis enzymes was tested using individual spectrophotometric assays for each enzyme. All four enzymes are active, and the activities in supernatants of the expression assays ranged from 2 to 10 units/mg of protein.

Using cell-free lysates from the strain overexpressing RmlA, dTDP-D-glucose was synthesized from glucose-1-P and dTTP. dTDP-6-deoxy-D-xylo-4-hexulose was synthesized from dTDP-D-glucose using RmlB, as expected from work reported by others (12, 13). The yield of dTDP-6-deoxy-D-xylo-4-hexulose was 100% after reaction. The product was purified by anion exchange high performance liquid chromatography on a CarboPac PA-1 column and the final yield after purification was approximately 95%. Following reduction and subsequent hydrolysis, 6-deoxyglucose and 6-deoxygalactose (fucose) were formed from the reaction product. Each component was identified by comparison with internal and external standards. The products are consistent with reduction of the 4-keto group of dTDP-6-deoxy-D-xylo-4-hexulose.

To prove the ability of RmlC to produce dTDP-6-deoxy-L-lyxo-4-hexulose, dTDP-D-glucose was reacted with RmlB and RmlC. The purified reaction mixture contained both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-6-deoxy-L-lyxo-4-hexulose in a ratio of approximately 97:3, as shown by a spectrofluorometric assay. Consistent with this result, radiolabeling of the 4-keto group with NaB[³H]₄, followed by hydrolysis of the labeled nucleotide-bound monosaccharides, resulted in two major and one minor radioactive peaks that could be separated by HPAEC/PED (Fig. 2). Using external and internal standards, the major peaks were identified as fucose and 6-deoxyglucose (products of dTDP-6-deoxy-D-xylo-4-hexulose), and the minor one as rhamnose (product of dTDP-6-deoxy-L-lyxo-4-hexulose). However, 6-deoxytalose, the second product of dTDP-6-deoxy-L-lyxo-4-hexulose, was not detected; this may coelute with one of the other monosaccharides.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   HPAEC/PED analysis of a reduced (NaB[3H]4) and hydrolyzed mixture of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-6-deoxy-L-lyxo-4-hexulose, synthesized by incubation of dTDP-D-glucose with RmlB and RmlC. Fractions (0.25 ml) were collected and analyzed for incorporated radioactivity. The major peaks represent fucose (1) and 6-deoxyglucose (2) derived from dTDP-6-deoxy-D-xylo-4-hexulose, the minor peak represents rhamnose (3) from dTDP-6-deoxy-L-lyxo-4-hexulose. The expected 6-deoxytalose product was not detected and probably coelutes with one of the other components. The arrows indicate maximum PED response of the respective monosaccharides. Rhamnose was detected by HPAEC/PED only when added as an internal standard (see inset).

For synthesis of dTDP-L-rhamnose, dTDP-D-glucose was reacted with RmlB, C, and D. For in situ regeneration of NADH, a formate dehydrogenase catalyzing the NAD⁺-dependent oxidation of formate was used (Fig. 3, track A). The yield of dTDP-L-rhamnose in this reaction was 100%. After removal of the proteins and salts the reaction mixture contained approximately 5% of NADH (Fig. 3, track B), and this was removed from the reaction mixture using anion exchange chromatography. Purified dTDP-L-rhamnose (yield 80%) contained no other nucleotide as evidenced by the single peak in HPAEC analysis on a CarboPac PA-1 column (Fig. 3, track C). Identity of dTDP-L-rhamnose was demonstrated by monosaccharide analysis of the hydrolyzed product.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   High performance liquid chromatography analysis of enzymatic synthesis of dTDP-L-rhamnose. A, plot showing substrates dTDP-D-glucose and NAD+ before addition of enzyme. B, reaction products obtained after addition of RmlB, RmlC, and RmlD to substrates and using formate dehydrogenase for regeneration of NADH. C, purified reaction product, dTDP-L-rhamnose.

To prove the substrate specificity of the three enzymes converting dTDP-D-glucose to dTDP-L-rhamnose (RmlB, RmlC, and RmlD), UDP-D-glucose, CDP-D-glucose, ADP-D-glucose, and GDP-D-glucose were used as substrates instead of dTDP-D-glucose. The activities with all these substrates were lower than 1% of the activity with dTDP-D-glucose.

Distinction between dTDP-4-dehydrorhamnose 3,5-Epimerase and dTDP-4-dehydrorhamnose Reductase-- The existing assignments of RmlC and RmlD to specific enzymatic activities are based on sequence data and there is some ambiguity in the literature. Direct assays for the separate measurement of RmlC activity or RmlD activity are not available. However, by using a coupled assay in which RmlC and RmlD are utilized together, and by varying the conditions in the assay, the activities of RmlC and RmlD were clearly distinguished. Quantitation in the assays is based on the consumption of NADPH by the reductase, which is conveniently monitored by a time-dependent decrease in the absorbance at 340 nm. When RmlD was used in 20-fold molar excess over RmlC, the initial reaction velocity of RmlC, determined with 0.18 mM dTDP-6-deoxy-D-xylo-4-hexulose, is not dependent on the NADPH concentration in a range of 0.007-0.109 mM. This result identified RmlC as the epimerase and RmlD as the reductase. Accordingly, with RmlC being used in 100-fold molar excess, the activity of RmlD can be specifically measured.

Conditions for Enzyme Assays and Synthesis of dTDP-activated Monosaccharides-- Under the assay conditions employed, RmlC shows maximum activity at pH 7.5 and is stable over a wide pH range. Its pH activity profile is similar to that of RmlD, indicating that their inclusion in a coupled RmlCD assay is not compromised by pH considerations. The pH optimum of RmlD is 6.5, but stability is highly dependent on the pH conditions. Even at pH 7.0 approximately 75% of RmlD activity is lost during incubation at 37 °C for 20 min. However, RmlD is more stable at temperatures below 30 °C (data not shown). Treatment with chelators such as EDTA (up to 0.3 mM) causes a reversible loss of up to 70% of enzyme activity for both RmlC and RmlD. Upon addition of MgCl₂ in concentrations exceeding that of EDTA full activity is restored.

Kinetic Constants for RmlC and Its Potential Interaction with RmlD-- For determination of RmlC activity, the assay was coupled with RmlD (20-fold molar excess) producing NADP⁺ from NADPH. The K_m value of RmlC for dTDP-6-deoxy-D-xylo-4-hexulose was 0.71 ± 0.17 mM and k_cat was 39 ± 6.5 s¹ (Table I). There is clear evidence for product formation from dTDP-6-deoxy-D-xylo-4-hexulose by RmlC acting in the absence of RmlD. However, dTDP-6-deoxy-L-lyxo-4-hexulose was extremely unstable, decomposing too rapidly to allow its isolation on a preparative scale. Therefore, no kinetic data were obtained for the reverse reaction of RmlC.

One explanation for the very low amount of dTDP-6-deoxy-L-lyxo-4-hexulose, if RmlC is examined in isolation, is that RmlC and RmlD form a complex, converting dTDP-6-deoxy-D-xylo-4-hexulose to dTDP-L-rhamnose. This was proposed in the early work of Melo and Glaser (30), in a scenario where only dTDP-6-deoxy-L-lyxo-4-hexulose bound to the enzyme complex can be reduced by the reductase. dTDP-6-deoxy-L-lyxo-4-hexulose would therefore not exist in free form. To address this hypothesis, mixtures of RmlC and RmlD, with and without substrate, were examined in nondenaturing anionic PAGE. No evidence was found for tight complexes of both enzymes, even in overloaded silver-stained gels (Fig. 4). Additionally, enzyme assays were performed in the presence of Ficoll. Ficoll can act as a macromolecular crowding agent to inhibit the diffusion of proteins (31, 32) and should strengthen the interaction of any macromolecular assemblies involving a dTDP-6-deoxy-L-lyxo-4-hexulose·RmlC complex. Enzyme activity was not influenced by addition of Ficoll at concentrations of up to 30%, making a complex of RmlC and RmlD, formed during catalysis, very unlikely.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Nondenaturing PAGE in Tris/citrate buffer. Lane 1, RmlC (1 µg); lane 2, RmlD (1 µg); lane 3, mixture of RmlC and RmlD; lane 4, mixture of RmlC, RmlD, and dTDP-6-deoxy-D-xylo-4-hexulose; lane 5, mixture of RmlC, RmlD, dTDP-6-deoxy-D-xylo-4-hexulose, and NADPH.

Kinetic Measurements for RmlD-- The pH optimum for the reduction of dTDP-6-deoxy-L-lyxo-4-hexulose by RmlD is 6.5. At pH values above 9.0 NAD(P)⁺-dependent oxidation of dTDP-L-rhamnose was detectable. The activity for the reverse reaction was less than 1% of the activity of the reduction reaction, with a maximum at pH 10.0. In the presence and absence of RmlC, similar initial reaction velocities were detected, although the equilibrium levels of NADH were different. The equilibrium concentration of substrates and products formed in the absence of RmlC allowed calculation of the K_eq,RmlD to be 3.6 × 10¹³ (± 1.5 × 10¹³, n = 6) in reaction 4. When RmlC was added, the calculated K_eq,RmlC × K_eq,RmlD value was 4.5 × 10¹¹ (± 1.1 × 10¹¹, n = 6). From these results K_{eq, RmlC} was estimated to be 0.013. This value is in agreement with the detection of low amounts of free dTDP-6-deoxy-L-lyxo-4-hexulose in equilibrium with dTDP-6-deoxy-D-xylo-4-hexulose.

Since isolation of dTDP-6-deoxy-L-lyxo-4-hexulose was not feasible due to instability of the product, this intermediate was generated in situ for kinetic analysis of RmlD using a 100-fold molar excess of RmlC. Apparent K_m and k_cat values for NADH and NADPH were determined with a constant concentration of dTDP-6-deoxy-D-xylo-4-hexulose (Table I). RmlD shows dual coenzyme specificity for NADH and NADPH with a slight preference for NADH. The initial velocities were determined with several fixed concentrations of dTDP-6-deoxy-D-xylo-4-hexulose and varying concentrations of NADPH. Double-reciprocal plots obtained from these data showed an intersecting pattern (not shown), indicating a sequential, ternary complex mechanism of RmlD. A fit of the experimental data to Equation 3 by nonlinear regression is shown in Fig. 5. The constants derived from this analysis were: K_m,NADH = 0.21 ± 0.004 mM, K_m,hexulose = 0.106 ± 0.018 mM, k_cat = 53 ± 4 s¹, and k_I = 0.012 ± 0.005 mM. At pH 7.0, no reverse reaction was detectable. Therefore, the dissociation constants for NAD⁺, NADP⁺, and dTDP-L-rhamnose were determined by fluorescence titration (_excitation at 280 nm and _emission at 350 nm). The addition of each component to a solution of RmlD (7.5 nM in 50 mM potassium phosphate buffer, pH 7.0) caused a significant quenching of the intrinsic tryptophan fluorescence of the enzyme. Scatchard analysis of the data allowed calculation of dissociation constants and determination of the numbers of binding sites for NAD⁺, NADP⁺, and dTDP-L-rhamnose. K_d values were 1.0, 2.3, and 0.37 mM, respectively, and one binding site was predicted for both NAD(P)⁺ and dTDP-L-rhamnose. When both NAD(P)⁺ and dTDP-L-rhamnose were used sequentially for fluorescence titration they showed additive quenching effects.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Reaction mechanism for RmlD. Dependence of RmlD activity on NADPH concentration was tested at different dTDP-6-deoxy-D-xylo-4-hexulose concentrations: 90 µM (), 180 µM (), 360 µM (). The lines drawn represent a fit of the experimental data to Equation 3.

RmlD Resembles the Reductase/Epimerase/Dehydrogenase (RED) Protein Superfamily-- The search for conserved motifs rather than overall sequence identity may reveal family relationships, even when overall primary sequence identity/similarity is not higher than 15-20%. To find conserved regions in RmlD sequences, a multiple sequence alignment was performed using sequences from 23 different bacteria and archaea (Fig. 6). The sequences used for Shigella flexneri 2a (33), Xanthomonas campestris (34), and Saccharopolyspora erythrea (35) have been described as dTDP-4-dehydrorhamnose 3,5-epimerases but are, according to sequence similarities, the corresponding reductases. Some of the sequences have not yet been definitively designated as RmlD or dTDP-4-dehydrorhamnose reductase, but have distinct homologies to the S. enterica LT2 RmlD sequence. All of the RmlD sequences contain a strictly conserved (Y-X₃-K) motif that is characteristic for the single domain reductase/epimerase/dehydrogenase protein superfamily (RED family, see Refs. 36 and 37). The RED family includes the short-chain dehydrogenases/reductases (38). The (Y-X₃-K) motif is located within a larger conserved domain, identified in Fig. 6 as "motif 3." Furthermore, the typical coenzyme-binding Rossman fold, containing a modified Wierenga motif (G-X₂-G-X₂-G), was identified previously in the amino-terminal part of the RmlD sequence (39). The same motif is present in all other members of this protein family. The crystal structures of two functionally related members of the RED family, UDP-galactose epimerase (40, 41) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (42, 43), have been elucidated recently. The active site of both enzymes was shown to consist of a catalytic triad of serine, tyrosine, and lysine (40, 43). The catalytic serine residue is located upstream of the Y-X₃-K loop. A conserved serine residue has also been found in the RmlD sequences, located in a highly conserved region labeled "motif 2" in Fig. 6. The Swiss Protein data base was searched for the sequence STDYVF, but no additional protein containing this motif was identified. Alignments of RmlD sequences with UDP-galactose epimerase and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase sequences revealed distant relationships (and limited similarity) among these proteins, but the catalytic amino acids are conserved in all of these proteins (not shown). As expected from differences in substrate and coenzyme specificitiy, residues known to be important for substrate or coenzyme-binding in UDP-galactose epimerase and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase were not conserved in RmlD. However, presence of the Rossman-fold and the catalytic center residues identify RmlD as a novel member of the RED protein superfamily.

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Multiple sequence alignment of 23 deduced RmlD amino acid sequences from different bacterial species. S. enterica LT2 (Swiss Protein P26392), S. flexneri 2a (PIR C55213), E. coli O7:K1 (Swiss Protein Q46769), E. coli K12 (Swiss Protein P37760), Burkholderia pseudomallei (EMBL AF064070), X. campestris (PIR C49906), Synechocystis sp. (DDBJ D90899), Sphingomonas S88 (GenBank U51197), Sinorhizobium meliloti (EMBL Z79692), Rhizobium sp.(Swiss Protein P55463), Azorhizobium caulinodans (PIR B36864), Actinobacillus actinomycetemcomitans (DDBJ AB010415), Serratia marcescens (GenBank AF038816), S. erythrea (GenBank L37354), M. tuberculosum (EMBL Z92771), Streptomyces glaucescens (EMBL AJ006985), Streptomyces griseus (Swiss Protein P29781), Streptococcus pneumoniae 19F (GenBank AF030362), Streptococcus mutans (DDBJ AB000631), Methanobacterium thermoautotrophicum (GenBank AE000933), Pyrococcus horikoshii (DDBJ AP000002), Bacillus subtilis (Swiss Protein P39631), L. interrogans (GenBank U61226). The Rossman fold (motif 1) is located upstream of motifs 2 and 3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

L-Rhamnose is a common constituent of different bacterial glycoconjugates. The biosynthetic precursor, dTDP-L-rhamnose, is formed by the sequential action of the RmlABCD enzymes. Detailed mechanisms have been proposed for synthesis of dTDP-D-glucose by RmlA (11) and its conversion to dTDP-6-deoxy-D-xylo-4-hexulose by RmlB (12). The final two steps are catalyzed by RmlC and RmlD and convert dTDP-6-deoxy-D-xylo-4-hexulose to dTDP-L-rhamnose. These activities are now assigned by the studies reported here.

Using purified enzymes, dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose were synthesized enzymatically from dTDP-D-glucose for further analyses of RmlC and RmlD. Although the intermediate product, dTDP-6-deoxy-L-lyxo-4-hexulose, was detected, it could not be isolated in significant amounts when synthesized either from dTDP-6-deoxy-D-xylo-4-hexulose with RmlC, or from dTDP-L-rhamnose in the reverse reaction of RmlD.

Melo and Glaser (30) postulated that the epimerase and reductase might form a complex, so that dTDP-6-deoxy-L-lyxo-4-hexulose is only reduced by RmlD when the substrate is bound to RmlC. From the experiments described here, we found no data consistent with such a complex. In addition, in a separate study using the E. coli mutant strain Y10, defective in RmlD, Wahl and Grisebach (44) reported that approximately 1% of ³H-labeled dTDP-6-deoxy-D-xylo-4-hexulose is converted to dTDP-6-deoxy-L-lyxo-4-hexulose. In agreement with these data, we detected conversion of about 3% of dTDP-6-deoxy-D-xylo-4-hexulose by RmlC from S. enterica LT2. The yield of free dTDP-6-deoxy-L-lyxo-4-hexulose is explained by the relatively small equilibrium constant for RmlC alone. In a coupled system comprising RmlC and RmlD, the reductase nature of RmlD enables efficient biosynthesis of dTDP-L-rhamnose. A similar biosynthetic pathway is known for the de novo biosynthesis of GDP-L-fucose. The last two steps in this pathway are catalyzed by a bifunctional epimerase/reductase (42, 43). Probably due the instability of the intermediate, strategies have evolved to keep its concentration low. In the case of dTDP-L-rhamnose, equilibrium concentration of dTDP-6-deoxy-L-lyxo-4-hexulose is indeed low, whereas for synthesis of GDP-L-fucose epimerization and reduction are catalyzed by the same active site (43). While the precise mechanism of action of RmlC is not yet known, crystals of purified S. enterica LT2 RmlC diffracting to 2.17 Å have recently been isolated (45). Preliminary analysis of the crystal data indicates a dimer structure, explaining the gel-filtration results presented here. Ultimately, solution of the RmlC structure will direct subsequent detailed analysis of the reaction mechanism.

RmlD has a dual coenzyme specificity and judging from the apparent K_m values, it binds both NADH and NADPH with high affinity. The kinetics of NADPH-dependent reduction of dTDP-6-deoxy-L-lyxo-4-hexulose by RmlD are consistent with a ternary complex mechanism of the enzyme. Since the RmlD reverse reaction could not be performed at pH 7.0, binding studies for NAD(P)⁺ and dTDP-L-rhamnose were performed using the quenching effect on protein fluorescence of these products. Binding constants are in the millimolar range, and NAD(P)⁺ and dTDP-L-rhamnose seem to bind independently to different sites.

In the common dehydrogenase/reductase signature (Y-X₃-K) one candidate active site amino acid is tyrosine (46). This signature was found in a multiple sequence alignment of 23 RmlD sequences (Fig. 6). An extended consensus region (motif 3) containing Y-X₃-K was identified in known and predicted RmlD homologues. In addition to this motif and the Rossman fold, including a Wierenga motif (G-X₂-G-X₂-G), another conserved region (motif 2) was found upstream of motif 3 (Fig. 6). This pattern is highly conserved. For example, an exchange of tyrosine to phenylalanine at position 7 of motif 2, or glycine to alanine at position 11, was only found in L. interrogans (5). Since the site for NAD(P)H binding (the Rossman fold) and the catalytic center (Y-X₃-K) are well defined within RmlD homologues, motif 2 may be involved in binding of dTDP-6-deoxy-L-lyxo-4-hexulose. The serine residue in motif 2 likely participates with the tyrosine and lysine residues in the Y-X₃-K motif, to form the catalytic serine-tyrosine-lysine triad identified in the crystal structures of UDP-galactose-4-epimerase (GalE) (40, 41) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER) (42, 43). Interestingly, a region resembling motif 2, with the sequence STDEVY, was also found in RmlB of S. enterica LT2. Notably, RmlB binds a substrate similar to that of RmlD, but catalyzes the oxidation of the hydroxyl group at C4.

The sequences used for S. flexneri 2a (33), X. campestris (34), and S. erythrea (35) have been described as dTDP-4-dehydrorhamnose 3,5-epimerases. Other sequences have not yet been definitively designated as dTDP-4-dehydrorhamnose reductase, dTDP-6-deoxymannose dehydrogenase, or dTDP-rhamnose synthetase, but according to sequence similarities they all are RmlD homologues. The consensus sequences identified here facilitate clarification of these enzyme activities. RmlD proteins should contain: (i) a Rossman fold in the NH₂-terminal region (motif 1); (ii) motif 2; and (iii) the conserved Y-X₃-K loop within an extended motif 3. Possession of motif 2 distinguishes RmlD from other members of the RED family. RmlD shows some similarities to GalE (40, 41) and GMER (42, 43). The structures of these proteins have been elucidated and allowed their assignment to the RED protein superfamily. RmlD shows homology to GalE and GMER in those amino acids known to have catalytic function, but GalE and GMER lack the distinctive motif 2 that characterizes RmlD homologues. In conclusion, RmlD is a novel member of the RED protein superfamily.

Kinetic analysis of RmlC and RmlD, including pH optima and requirement of specific ions led to the development of a novel method for synthesis of dTDP-L-rhamnose from dTDP-D-glucose. Highly efficient enzymatic synthesis of dTDP-L-rhamnose was achieved with a mixture of RmlB, RmlC, RmlD, and by including an internal system to recycle NAD⁺ to NADH (Fig. 3). After removal of the proteins and salts, the reaction supernatant contained approximately 5% of NADH, which should not be inhibitory for most of the reactions dTDP-L-rhamnose is used for (e.g. rhamnosyltransferases). Following purification, the yield of dTDP-L-rhamnose was 80%. Such a reaction system is ideal for generating substrates for studies of rhamnosyltransferase activities and the use of these enzymes for enzymatic synthesis of complex carbohydrates.

	ACKNOWLEDGEMENTS

P. M. thanks all colleagues in the laboratory of C. Whitfield, in particular Paul A. Amor, for tremendous support during his stay in Guelph.

	Addendum

While this paper was in the final stages of the review process, data for the action of RmlC in E. coli and Mycobacterium tuberculosis was published by another research group (47). Their findings are consistent with the results described here.

	FOOTNOTES

^* This work was supported by Austrian Science Fund Grants S7201-MOB and P12966-MOB (to P. M.), the Austrian Ministry of Science and Transportation, and the Natural Sciences and Engineering Research Council and the Canadian Bacterial Diseases Network (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by postdoctoral fellowships from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council.

^** To whom correspondence should be addressed: Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Gregor-Mendel-Str. 33, A-1180 Wien, Austria. Tel.: 43-1-476-54 (ext. 2202); Fax: 43-1-478-91-12; E-mail: pmessner@edv1.boku.ac.at.

	ABBREVIATIONS

The abbreviations used are: dTDP, thymidine diphosphate; RmlA, glucose-1-phosphate thymidyltransferase; RmlB, dTDP-D-glucose 4,6-dehydratase; RmlC, dTDP-4-dehydrorhamnose 3,5-epimerase; RmlD, dTDP-4-dehydrorhamnose reductase; HPAEC/PED, high performance anion exchange chromatography-pulsed electrochemical detection; PAGE, polyacrylamide gel electrophoresis; LB, Luria-Bertani; RED family, reductase/epimerase/dehydrogenase protein superfamily; GMER, GDP-4-keto-6-deoxy-D-mannose epimerase/reductase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES