Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase

Abstract

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[␣-D-U-14 C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic ␣-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDPgalactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 ؎ 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

UDP-galactofuranose Precursor Required for Formation of the Lipopolysaccharide O Antigen of Klebsiella pneumoniae Serotype O1 Is Synthesized by the Product of the rfbDKPO1 Gene

“…6C), while no reaction was observed in assays lacking a cofactor or in assays with NAD or NADP (data not shown). Other than the cofactor requirements, these data are in agreement with the data de- scribed for Glf of E. coli (48) and further support the conclusion that p37 indeed is UDP-Galf.…”

“…Based on chemical analysis of the reaction products obtained by acid and alkaline hydrolosis as well as periodate oxidation, Trejo et al (46) predicted that the sugar nucleotide is UDP-Galf. More recently, Nassau et al (48) cloned the glf gene of E. coli and showed that it encodes UDP-galactopyranose mutase (EC 5.4.99.9). Nassau et al (48) concluded that the reaction product of the UDP-galactopyranose mutase reaction is UDP-Galf based on HPLC analysis of the sugar 1-phosphate obtained after phosphodiesterase treatment of the reaction product.…”

“…More recently, Nassau et al (48) cloned the glf gene of E. coli and showed that it encodes UDP-galactopyranose mutase (EC 5.4.99.9). Nassau et al (48) concluded that the reaction product of the UDP-galactopyranose mutase reaction is UDP-Galf based on HPLC analysis of the sugar 1-phosphate obtained after phosphodiesterase treatment of the reaction product. The modification of an HPAEC procedure, to give a method that separates UDP-Galp from UDP-Galf, allowed us for the first time to purify milligram quantities of UDP-Galf and to unequivocally prove by NMR the structure to be uridine 5Ј-(trihydrogen diphosphate) PЈ-␣-D-galactofuranosyl ester.…”

“…The Galf precursor for a galactofuranosyl-containing polysaccharide in Penicillium charlesii was also proposed to be a UDP-linked derivative (46). More recently, the product of orf6, an enzyme encoded by the cryptic rfb locus in E. coli K-12 strains (47), was shown to have UDP-galactopyranose mutase (EC 5.4.99.9) activity, capable of the reversible formation of UDP-Galf from UDP-Galp (48). The product UDP-Galf was predicted from degradation products and this compound has not been isolated and fully characterized in an intact state.…”

Relevant glycoconjugates on the surface of Trypanosoma cruzi

“…or (4) alternatively, would the latter be incorporated into a macromolecule via a UDP-Gal f precursor? Nassau et al (1996), have been able to obtain clones of UDP-galactopyranose mutase from E. coli K-12 capable to convert reversibly UDP-gal p to UDP-gal f with the equilibrium favoring the formation of the pyranoic compound. More recently, mutases have been cloned in Klebsiella pneumoniae (Koplin et al 1997) and Mycobacteria (Weston et al 1998).…”