dTDP-rhamnose is made from glucose-1-phosphate and dTTP by four enzymes encoded by An 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 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 gene was expressed and incubation of the resulting purified 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. Thus the 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 configuration.


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