Conversion of dTDP-4-keto-6-deoxyglucose to free dTDP-4-keto-rhamnose by the rmIC gene products of Escherichia coli and Mycobacterium tuberculosis.
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.
Stern
Richard J
RJ
Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
Lee
Tae-Yoon
TY
Department of Microbiology, College of Medicine, Yeungnam University, 317-1 Daemyung-5-dong, Namku, Taegu 705-305, Korea.
Lee
Tae-Jin
TJ
Department of Microbiology, College of Medicine, Yeungnam University, 317-1 Daemyung-5-dong, Namku, Taegu 705-305, Korea.
Yan
Wenxin
W
Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
Scherman
Michael S
MS
Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
Vissa
Varalakshmi D
VD
Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
Kim
Soo-Ki
SK
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Wanner
Barry L
BL
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
McNeil
Michael R
MR
Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
D002238:Carbohydrate Epimerases / Q000378:metabolism*; D003001:Cloning, Molecular; D017666:Deuterium Oxide / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D017353:Gene Deletion; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis; D012210:Rhamnose / Q000031:analogs & derivatives* / Q000378:metabolism; D013942:Thymine Nucleotides / Q000096:biosynthesis / Q000378:metabolism*; D001426:Bacterial Proteins; D002237:Carbohydrate Dehydrogenases
D002238:Carbohydrate Epimerases; D017666:Deuterium Oxide; D005947:Glucose; D009702:Nucleoside Diphosphate Sugars; D012210:Rhamnose; D013942:Thymine Nucleotides; C119260:dTDP-4-ketorhamnose; C112447:dTDP-6-deoxy-4-ketoglucose; C103522:deoxythymidine diphosphate-glucose; C073293:thymidine diphosphate rhamnose; C020092:dTDP-4-ketorhamnose 3,5-epimerase; D001426:Bacterial Proteins; D002237:Carbohydrate Dehydrogenases