UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype 01 is synthesized by the product of the rfbD(KP01) gene

The O-side-chain polysaccharide in the Lipopolysaccharide of Klebsiella pneumoniae O1 is based on a backbone structure of repeat units of [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; this structure is termed D-galactan I. The rfb (O-antigen biosynthesis) gene cluster directs the synthesis of D-galactan I and consists of six genes termed rfbA-F-KPO1. In this paper we show that rfbD(KPO1) encodes a UDP-galactopyranose mutase (NAD(P)H-requiring) (EC 5.4.99.9), which forms uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactofuranosyl ester (UDP-Galf), the biosynthetic precursor of galactofuranosyl residues. The deduced amino acid sequence of rfbD(KPO1) shows 85% and 37.5% identity to the rfbD(KPO8) gene of K. pneumoniae serotype O8 and the glf gene of Escherichia coli, respectively. The molecular mass of the purified RfbD(KPO1) enzyme is 45 kDa as determined by SDS-polyacrylamide gel electrophoresis, while gel filtration revealed a molecular mass of 92 kDa, suggesting a dimeric structure for the native protein. The rfbD(KPO1) gene product interconverts uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactopyranosyl ester (UDP-Galp) and UDP-Galf: Unlike Glf, RfbD(KPO1) showed a requirement for NADH or NADPH, which could not be replaced by NAD or NADP. RfbD(KPO1) was used to synthesize milligram quantities of UDP-Galf, allowing this compound to be purified and fully characterized in an intact form for the first time. The structure of UDP-Galf was proven by NMR spectroscopy.