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

• Published in: Microbiology (Society for General Microbiology) Vol. 145; no. 3; pp. 663 - 671
• Main Authors: Stern, Richard J, Lee, Tae-Yoon, Lee, Tae-Jin, Yan, Wenxin, Scherman, Michael S, Vissa, Varalakshmi D, Kim, Soo-Ki, Wanner, Barry L, McNeil, Michael R
• Format: Journal Article
• Language: English
• Published: Soc General Microbiol 01-03-1999
• Subjects: Escherichia coli; Mycobacterium tuberculosis
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-145-3-663

1 Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA
2 Department of Microbiology, College of Medicine, Yeungnam University, 317-1 Daemyung-5-dong, Namku, Taegu 705-305, Korea
3 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
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ABSTRACT
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. Thus 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.
Keywords: rhamnose, rmlC, mycobacterial cell wall, drug development, dTDP-4-keto-6-deoxyglucose epimerase