Cloning and expression of the glucose-1-phosphate thymidylyltransferase gene (gerD) from Streptomyces sp. GERI-155

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules, that has antimicrobial activity against Gram-positive bacteria. The deoxysugar biosynthetic gene cluster of GERI-155 was cloned from Streptomyces sp., GERI-155. One of the orfs, gerD, appeared to encode glucose-1-phosphate thymidylyltransferase (dTDP-glucose synthase), which converts dTTP and glucose-1-phosphate to dTDP-D-glucose and pyrophosphate. GerD was expressed in E. coli in vector pHJ2 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and DEAE-Sepharose CL-6B and DEAE-Trisacryl column chromatography. The specific activity of the enzyme increased 16-fold with a recovery of 10%. It migrated as a single band on SDS-PAGE with a molecular mass of 30 kDa. The purified protein had glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction the highest activity was obtained with the combination of dTTP and alpha-D-glucose-1-phosphate, and only 5.5% of that activity was obtained with UTP in place of dTTP. In the opposite direction the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.     

The molecular architecture of glucose-1-phosphate uridylyltransferase

Glucose-1-phosphate uridylyltransferase, also referred to as UDP-glucose pyrophosphorylase or UGPase, catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP. Not surprisingly, given the central role of UDP-glucose in glycogen synthesis and in the production of glycolipids, glycoproteins, and proteoglycans, the enzyme is ubiquitous in nature. Interestingly, however, the prokaryotic and eukaryotic forms of the enzyme are unrelated in amino acid sequence and structure. Here we describe the cloning and structural analysis to 1.9 A resolution of the UGPase from Escherichia coli. The protein is a tetramer with 222 point group symmetry. Each subunit of the tetramer is dominated by an eight-stranded mixed beta-sheet. There are two additional layers of beta-sheet (two and three strands) and 10 alpha-helices. The overall fold of the molecule is remarkably similar to that observed for glucose-1-phosphate thymidylyltransferase in complex with its product, dTDP-glucose. On the basis of this similarity, a UDP-glucose moiety has been positioned into the active site of UGPase. This protein/product model predicts that the side chains of Gln 109 and Asp 137, respectively, serve to anchor the uracil ring and the ribose of UDP-glucose to the protein. The beta-phosphoryl group of the product is predicted to lie within hydrogen bonding distance to the epsilon-nitrogen of Lys 202 whereas the carboxylate group of Glu 201 is predicted to bridge the 2'- and 3'-hydroxyl groups of the glucosyl moiety. Details concerning the overall structure of UGPase and a comparison with glucose-1-phosphate thymidylyltransferase are presented.     

Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7

L-rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of L-rhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100 degrees C, with a half-life of 60 min at 95 degrees C and 180 min at 80 degrees C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing alpha-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not alpha-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

The complex of Sphingomonas elodea ATCC 31461 glucose-1-phosphate uridylyltransferase with glucose-1-phosphate reveals a novel quaternary structure, unique among nucleoside diphosphate-sugar pyrophosphorylase members

Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.     

Biosynthesis of enterobacterial common antigen requires dTDPglucose pyrophosphorylase determined by a Salmonella typhimurium rfb gene and a Salmonella montevideo rfe gene.

In group C1 salmonellae, rfe and rff genes linked to the ilv locus specify the synthesis of a glycolipid called the enterobacterial common antigen. In contrast, in group B salmonellae the synthesis requires in addition some of the genes in the rfb cluster, the main genetic determinant of the O side chains of lipopolysaccharide. In an effort to define the biochemical functions of these rfb genes, we looked in Salmonella typhimurium LT2 (group B) for rfb mutants in which the synthesis of both enterobacterial common antigen and the O side chains would be blocked in a manner suppressible by the wild-type rfe cluster of S. montevideo, of group C1. We found one mutant with these characteristics. This rfb mutation affected the activity of dTDPglucose pyrophosphorylase (glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24). Whereas the rfe cluster of S. montevideo contained a gene producing this enzyme activity, there was no evidence for the presence of such a gene in the rfe cluster of group B strains. These results also showed that the synthesis of dTDP-glucose is necessary for the biosynthesis of enterobacterial common antigen; this conclusion fits with the recent demonstration of 4-acetamido-4,6-dideoxy-D-galactose as a component of enterobacterial common antigen (Lugowski et al., Carbohydr. Res. 118:173-181, 1983), because the biosynthesis of the donor of this sugar, dTDP-4-acetamido-4,6-dideoxy-D-galactose, requires dTDPglucose pyrophosphorylase.     

Proteins encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG genes, involved in gellan gum biosynthesis, exhibit both dTDP- and UDP-glucose pyrophosphorylase activities

The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 microM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 microM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).     

Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.

We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.     

Cloning and expression of glucose-1-phosphate thymidylyltransferase gene (schS6) from Streptomyces sp. SCC-2136

The deoxysugar biosynthetic gene cluster of Sch 47554/Sch 47555 was cloned from Streptomyces sp. SCC-2136. One of the ORFs, schS6, appeared to encode glucose-1-phosphate thymidylyltransferase, which converts dTTP and glucose-1-phosphate to TDP-D-glucose and pyrophosphate. The dTDP-D-glucose is a key metabolite in prokaryotics as a precursor for a large number of modified deoxysugars, and these deoxysugars are a major part of various antibiotics, ranging from glycosides to macrolides. SchS6 was expressed in E. coli vector pSCHS6 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and Ni-NTA affinity column chromatography. The specific activity of the purified enzyme increased 4.7-fold with 17.5% recovery. It migrated as a single band on SDS-PAGE with an apparent molecular mass of 56 kDa. The purified protein showed glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction, the highest activity was obtained with combination of dTTP and alpha-D-glucose-1-phosphate, and only 12% of that activity was obtained with the substrates UTP/alpha-D-glucose-1-phosphate. In the opposite direction, the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.     

Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies, NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP+ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903-904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP+] ratio.     

Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster: identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene.

The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N-acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster.     

Cloning, expression and characterization of glucose-1-phosphate thymidylyltransferase (strmlA) from Thermus caldophilus

dTDP-L-Rhamnose biosynthetic gene cluster was cloned from Thermus caldophilus. A cluster of four open reading frames, strmlA, B, C and D, responsible for the production of dTDP-L-rhmanose, was screened from the genomic library. Thermophilic glucose-1-phosphate thymidylyltransferase, encoding 356 amino acids with a calculated molecular weight 38 kDa, was expressed under the control of the tac promoter in E. coli. The expressed enzyme, stRmlA is thermostable up to 70 degrees C and apparently retained its activity even up to 90 degrees C. It shares 73% sequence identity to glucose-1-phosphate thymidylyltransferase from Streptomyces argillaceus. Amino acid sequence comparison of stRmlA with ten glucose-1-phosphate thymidylyltransferases indicated higher number of unusual hydrophobic residues (A10, A58, A69, A252, V225, V257, V265, 1242 and 1246) and charged residues (D43, D160, D248, D249, D315, H124, H201, H283 and H354) in stRmlA.     

One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate

An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.     

Glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes. Kinetic mechanism and competitive substrate inhibition by glyceraldehyde 3-phosphate

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was isolated from human erythrocyte ghosts by a simple procedure utilizing ammonium sulfate precipitation and affinity chromatography on NAD⁺-Sepharose 4B. The purified enzyme had a specific activity of 98 units/mg protein. The kinetic mechanism of GAPD was studied by product and deadend inhibition using NADH, α-glycerophosphate, nitrate, and 2,3-diphosphoglycerate. The results indicated that the human erythrocyte GAPD-catalyzed reaction follows an ordered ter bi mechanism characterized by the sequential addition of NAD⁺, glyceraldehyde 3-phosphate (GAP), and phosphate to the enzyme and the sequential release of 1,3-diphosphoglycerate and NADH from the enzyme. This contrasts with the mechanism (rapid equilibrium random ter bi) proposed by Oguchi (1970, J. Biochem. (Tokyo)68, 427–439) who based his conclusion on the initial rate data alone. Since the Michaelis-Menten kinetics were not applicable to this enzyme because of the competitive substrate inhibition by GAP, we devised a new kinetic approach for determining the parameters of the GAPD-catalyzed reaction. Results of this study indicate that the GAPD-catalyzed reaction is regulated by both ATP and GAP. We propose that GAP acts as an “amplifier” for the feedback inhibition effect of ATP. We discuss the effect this may have played in causing controversy over the regulatory role of this enzyme in glycolysis.     

Kinetic properties of normal human erythrocyte glucose-6-phosphate dehydrogenase dimers.

The steady-state kinetics of normal human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) dimers were studied as a function of pH and temperature. Inhibition studies using glucosamine 6-phosphate, NADPH and p-hydroxymercuribenzoate (P-OHMB) were also carried out at pH 8.0. The existence of two binding sites on the enzyme with a transition from low to high affinity for NADP+ when NADP+ concentration is increased is indicated by the nonlinear Lineweaver-Burk plots and sigmoid kinetic patterns. NADPH inhibition was found to be competitive with respect to NADP+ and non-competitive with respect to glucose-6-phosphate. Logarithmic plot of Vmax against pH and inactivation by P-OHMB indicate the participation in the reaction mechanism of imidazolium group of histidine and sulhydryl groups. The initial velocity and product inhibition data gave results which are consistent with the dimeric enzyme following an ordered sequential mechanism. A possible random mechanism is ruled out by the inhibition results of glucosamine 6-phosphate.     

Kinetic studies of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: pH variation of kinetic parameters

The specificity and kinetic parameters of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been examined under a range of conditions in order to elucidate details about the mechanism of action of this enzyme. The rate of oxidation of glucose 6-phosphate is inhibited by the addition of various organic solvents. However, the low, inherent glucose dehydrogenase activity of this enzyme was stimulated under these conditions, and was further activated by divalent anions that were observed to be inhibitors of the glucose 6-phosphate dehydrogenation. From an examination of the pH variation of the enzyme kinetic parameters two groups on the enzyme that appear to be involved in the binding of the phosphate group of the sugar substrate have been detected. An enzyme catalytic group, probably a carboxylic acid, has been identified that accepts the proton from the hydroxyl group at carbon-1 of the sugar substrate during its oxidation to a lactone. The ionization of a group on the enzyme with a pK of 8.7 resulted in an increase in the maximum velocity of the glucose-6-phosphate dehydrogenase activity of the enzyme as a consequence of a pH-dependent product release step that is no longer rate limiting at high pH. Stabilization of gluconic acid-delta-lactone against nonenzymatic hydrolysis by organic solvents has allowed the kinetic parameters of the reverse reaction to be reliably measured for the first time in a narrow pH range.     

Rat liver cytoplasmic glucose-6-phosphate dehydrogenase. Steady-state kinetic properties and circular dichroism.

Steady-state kinetic studies including initial velocity, NADPH product inhibition, dead-end inhibition, and combined dead-end and product inhibition measurements with purified rat liver glucose-6-phosphate dehydrogenase indicate a sequential and obligatory addition of substrates in the order of NADP+, glucose-6-P for the catalytic pathway at pH 8.0. Although instability of 6-phosphoglucono-delta-lactone precluded product inhibition experiments which might directly exclude an enzyme-6-phosphoglucono-delta-lactone complex, the absence of an enzyme-glucose-6-P complex suggests that the enzyme-lactone product is unlikely and the release of products is also ordered, with NADPH released last. Consideration of the kinetic constants (Ka = 2.0 muM, Kiq = 13 muM) and cellular concentration of the substrates and products suggests extensive inhibition of the enzyme in vivo and control by the NADPH/NADP+ ratios. Circular dichroism spectra of the enzyme in 20 mM phosphate buffer at pH 7.0 and 25 degrees C indicate 51% helix and 33% pleated sheet structures which is considerably different from results (14% helix) with yeast enzymes.     

Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies,NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP⁺ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903–904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP⁺] ratio.     

Studies on the sucrose phosphate synthetase kinetic mechanism

The kinetic properties of wheat germ sucrose phosphate synthetase, which catalyzes the reaction UDP-glucose + fructose 6-phosphate → UDP + sucrose 6-phosphate have been studied. A plot of the reciprocal initial velocity versus reciprocal substrate concentration gave a series of intersecting lines indicating a sequential mechanism. Product inhibition studies showed that UDP was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. A dead-end inhibitor, inorganic phosphate, was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. The results of initial velocity and product and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.     

Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-beta-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155

The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of L-rhamnose- and D-glycero-D-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-L-rhamnose (dTDP-L-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-L-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91(T) produces D-rhamnose- and 3-acetamido-3,6-dideoxy-D-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91(T) for L-rhamnose and D-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91(T) is not able to synthesize dTDP-L-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.