title: Characterization of glycosynthase mutants derived from glycoside hydrolase family 10 xylanases.
abstract: Four xylanases belonging to glycoside hydrolase family 10-Thermotoga maritima XylB (TM), Clostridium stercorarium XynB (CS), Bacillus halodurans XynA (BH), and Cellulomonas fimi Cex (CF)-were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric beta-1,4-linked xylopyranose as a precipitate during reaction with alpha-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X(2) as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.
authors.affiliation: Enzyme Laboratory, National Food Research Institute, Ibaraki, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sugimura
authors.forename: Mamoru
authors.initials: M
authors.lastname: Nishimoto
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D014994:Xylose / Q000031:analogs & derivatives / Q000096:biosynthesis; D014995:Xylosidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: C431715:xylopyranose; D014994:Xylose; D006026:Glycoside Hydrolases; D014995:Xylosidases