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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 Km 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.