title: Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium.
abstract: Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes xanthosine by xanthosine phosphorylase (xapA), Salmonella cannot grow on xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (k(cat)/Km=1 to 4 x 10(4) M(-1)s(-1)). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (k(cat)/Km=0.7 to 1.2 x 10(3) M(-1)s(-1)) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and xanthosine and the hydrolytic capacity (k(cat)/Km) is 5-fold higher for xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of xanthosine.
authors.affiliation: Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83 H, 1307 Copenhagen K, Denmark.
authors.forename: Michael Riis
authors.initials: MR
authors.lastname: Hansen
authors.forename: Gert
authors.initials: G
authors.lastname: Dandanell
mesh_terms: D000241:Adenosine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007288:Inosine / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012263:Ribonucleosides / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity; D014529:Uridine / Q000378:metabolism*; D014970:Xanthines
chemical_list: D012263:Ribonucleosides; D014970:Xanthines; D007288:Inosine; C005893:xanthosine; D009699:N-Glycosyl Hydrolases; D000241:Adenosine; D014529:Uridine