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 a ssembly 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 react ions, 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-depend ent 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 b y showing its activity to be NAD(P)H-independent. Spectrofluorometric and r adiolabeling experiments were used to demonstrate the ability of RmlC to ca talyze 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 thym idine diphosphate-4-dehydrorhamnose reductase, The reductase property of Rm lD was shown by equilibrium analysis and its ability to enable efficient bi osynthesis 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 sequen ces identified several conserved amino acid residues, especially the serine -tyrosine-lysine catalytic triad, characteristic for members of the reducta se/epimerase/dehydrogenase protein superfamily, In conclusion, RmlD is a no vel member of this protein superfamily.