Volume 272, Number 7, Issue of February 14, 1997 pp. 4121-4128
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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UDP-galactofuranose Precursor Required for Formation of the Lipopolysaccharide O Antigen of Klebsiella pneumoniae Serotype O1 Is Synthesized by the Product of the rfbD_KPO1 Gene

(Received for publication, September 16, 1996, and in revised form, November 11, 1996)

Reinhard Köplin , Jean-Robert Brisson ^§ and Chris Whitfield

From the Canadian Bacterial Diseases Network,  Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1, and the ^§ Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada, K1A 0R6

The O-side-chain polysaccharide in the lipopolysaccharide of Klebsiella pneumoniae O1 is based on a backbone structure of repeat units of [3)--D-Galf-(13)--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--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--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.

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