Characterization of the dTDP-D-fucofuranose biosynthetic pathway in Escherichia coli O52.
D-fucofuranose (D-Fucf) is a component of Escherichia coli O52 O antigen. This uncommon sugar is also the sugar moiety of the anticancer drug--gilvocarcin V produced by many streptomycetes. In E. coli O52, rmlA, rmlB, fcf1 and fcf2 were proposed in a previous study by our group to encode the enzymes of the dTDP-D-Fucf (the nucleotide-activated form of D-Fucf) biosynthetic pathway. In this study, Fcf1 and Fcf2 from E. coli O52 were expressed, purified and assayed for their respective activities. Novel product peaks from enzyme-substrate reactions were detected by capillary electrophoresis and the structures of the product compounds were elucidated by electro-spray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Fcf1 was confirmed to be a dTDP-6-deoxy-D-xylo-hex-4-ulopyranose reductase for the conversion of dTDP-6-deoxy-D-xylo-hex-4-ulopyranose to dTDP-D-fucopyranose (dTDP-D-Fucp), and Fcf2 a dTDP-D-Fucp mutase for the conversion of dTDP-D-Fucp to dTDP-D-Fucf. The K(m) of Fcf1 for dTDP-6-deoxy-D-xylo-hex-4-ulopyranose was determined to be 0.38 mM, and of Fcf2 for dTDP-D-Fucp to be 3.43 mM. The functional role of fcf1 and fcf2 in the biosynthesis of E. coli O52 O antigen were confirmed by mutation and complementation tests. This is the first time that the biosynthetic pathway of dTDP-D-Fucf has been fully characterized.
Wang
Quan
Q
TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, PR China.
Ding
Peng
P
Perepelov
Andrei V
AV
Xu
Yanli
Y
Wang
Ying
Y
Knirel
Yuriy A
YA
Wang
Lei
L
Feng
Lu
L
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D009154:Mutation; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D019081:O Antigens / Q000096:biosynthesis* / Q000235:genetics; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D009702:Nucleoside Diphosphate Sugars; D019081:O Antigens; D013401:Sugar Alcohol Dehydrogenases