Conversion of dTDP-4-keto-6-deoxyglucase to free dTDP-4-keto-rhamnose by the rmlC gene products of Escherichia coli and Mycobacterium tuberculosis

dTDP-rhamnose is made from glucose-1-phosphate and dTTP by four enzymes enc oded by rmlA-D. An Escherichia coli rmlC mutant was constructed and a crude enzyme extract prepared from it did not produce dTDP-4-keto-rhamnose. in c ontrast to a crude enzyme extract prepared from a wild-type E. coli strain where small amounts of this intermediate were found after incubation with d TDP-glucose in the absence of NADPH. These results showed that dTDP-4-keto- rhamnose, the product of RmlC, exists as a free intermediate. Further, the Mycobacterium tuberculosis rmlC gene was expressed and incubation of the re sulting purified M. tuberculosis RmlC enzyme with dTDP-4-keto-6-deoxyglucos e resulted in the conversion of approximately 7% of dTDP-4-keto-6-deoxygluc ose to dTDP-4-keto-rhamnose. The enzyme also allowed for the incorporation of two deuterium atoms from deuterium oxide solvent into dTDP-4-keto-glucos e. Th us the rmlC gene encodes dTDP-4-keto-6-deoxyglucose epimerase capable of epimerizing at both C-3' and C-5'; this enzyme produces free dTDP-4-ket o-rhamnose but the equilibrium of the 4-keto sugar nucleotides lies strongl y on the side of the gluco configuration.