title: The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose.
abstract: We report the functional characterization of the galF gene of strain VW187 (Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PPi. We show that, although the GalF protein is expressed in vivo, GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF-/galU+ and galF/galU+ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo. We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, Canada.
authors.forename: C L
authors.initials: CL
authors.lastname: Marolda
authors.forename: M A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D016296:Mutagenesis; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose