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.
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.
Graninger
M
M
Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria.
Nidetzky
B
B
Heinrichs
D E
DE
Whitfield
C
C
Messner
P
P
D000595:Amino Acid Sequence; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis* / Q000378:metabolism; D019779:Salmonella enterica / Q000201:enzymology*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013942:Thymine Nucleotides / Q000096:biosynthesis* / Q000378:metabolism; D001426:Bacterial Proteins
D002237:Carbohydrate Dehydrogenases; D002238:Carbohydrate Epimerases; D009249:NADP; D009702:Nucleoside Diphosphate Sugars; D013942:Thymine Nucleotides; C073293:thymidine diphosphate rhamnose; C073295:thymidine diphosphate-6-deoxy-xylo-4-hexulose; C107144:dTDP-4-dehydrorhamnose reductase; C020092:dTDP-4-ketorhamnose 3,5-epimerase; D001426:Bacterial Proteins