microbiology, Vol 145, 663-671, Copyright © 1999 by Society for General Microbiology

ARTICLES

Conversion of dTDP-4-keto-6-deoxyglucose to free dTDP-4-keto-rhamnose by the rmIC gene products of Escherichia coli and Mycobacterium tuberculosis [In Process Citation]

RJ Stern, TY Lee, TJ Lee, W Yan, MS Scherman, VD Vissa, SK Kim, BL Wanner and MR McNeil
Department of Microbiology, Colorado State University, Fort Collins 80523, USA.

dTDP-rhamnose is made from glucose-1-phosphate and dTTP by four enzymes encoded by rmIA-D. An Escherichia coli rmIC 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 RmIC, exists as a free intermediate. Further, the Mycobacterium tuberculosis rmIC gene was expressed and incubation of the resulting purified M. tuberculosis RmIC 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 rmIC 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.

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