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 Klebsiell a 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 t he synthesis of D-galactan I and consists of six genes termed rfbA-F-K PO1. In this paper we show that rfbD(KPO1) encodes a UDP-galactopyrano se mutase (NAD(P)H-requiring) (EC 5.4.99.9), which forms uridine 5'-(t rihydrogen diphosphate) P'-alpha-D-galactofuranosyl ester (UDP-Galf), the biosynthetic precursor of galactofuranosyl residues. The deduced a mino acid sequence of rfbD(KPO1) shows 85% and 37.5% identity to the r fbD(KPO8) gene of K. pneumoniae serotype O8 and the glf gene of Escher ichia coli, respectively. The molecular mass of the purified RfbD(KPO1 ) enzyme is 45 kDa as determined by SDS-polyacrylamide gel electrophor esis, while gel filtration revealed a molecular mass of 92 kDa, sugges ting a dimeric structure for the native protein. The rfbD(KPO1) gene p roduct interconverts uridine 5'-(trihydrogen diphosphate) P'-alpha-D-g alactopyranosyl 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 o f UDP-Galf, allowing this compound to be purified and fully characteri zed in an intact form for the first time. The structure of UDP-Galf wa s proven by NMR spectroscopy.