title: Amino acid substitutions of His296 alter the catalytic properties of Zymomonas mobilis 10232 levansucrase.
abstract: His296 of Zymomonas mobilis levansucrase (EC 2.4.1.10) is crucial for the catalysis of the transfructosylation reaction. The three-dimensional structures of levansucrases revealed the His296 is involved in the substrate recognition and binding. In this study, nine mutants were created by site-directed mutagenesis, in which His296 was substituted with amino acids of different polarity, charge and length. The substitutions of His296 with Arg or Trp retained partial hydrolysis and transfructosylation activities. The positively charged Lys substitution resulted in a 2.5-fold increase of sucrose hydrolysis. Substitutions with short (Cys or Ser), negatively charged (Glu) or polar (Tyr) amino acids virtually abolished both the activities. Analysis of transfructosylation products indicated that the mutants synthesized different oligosaccharides, suggesting that amino acid substitutions of His296 strongly affected both the enzyme activity and transfructosylation products.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, PR China.
authors.forename: Shu Ying
authors.initials: SY
authors.lastname: Li
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.forename: Yong Liang
authors.initials: YL
authors.lastname: Yan
authors.forename: Hai Ying
authors.initials: HY
authors.lastname: Yu
authors.forename: Yu Hua
authors.initials: YH
authors.lastname: Zhan
authors.forename: Zi Xin
authors.initials: ZX
authors.lastname: Peng
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Lin
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D006602:Hexosyltransferases / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation; D013395:Sucrose / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D017355:Zymomonas / Q000201:enzymology*
chemical_list: D009841:Oligonucleotides; D009844:Oligosaccharides; D006639:Histidine; D013395:Sucrose; D014364:Tryptophan; D001120:Arginine; D006602:Hexosyltransferases; C021513:levansucrase