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 encoded 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 contrast to a crude enzyme extract prepared from a wild-type E. coli strain where small amounts of this intermediate were found after incubation with dTDP-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 resulting purified M. tuberculosis RmlC enzyme with dTDP-4-keto-6-deoxyglucose resulted in the conversion of approximately 7% of dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-rhamnose. The enzyme also allowed for the incorporation of two deuterium atoms from deuterium oxide solvent into dTDP-4-keto-glucose. 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-keto-rhamnose but the equilibrium of the 4-keto sugar nucleotides lies strongly on the side of the gluco configuration.