Two glutamic acids in chitosanase A from Matsuebacter chitosanotabidus 3001 are the catalytically important residues.
Chitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant k(cat) for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)(6) was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.
Shimono
Kumiko
K
Department of Life Science and Biotechnology, Faculty of Life & Environmental Science, and Research Institute of Molecular Genetics, Shimane University, Matsue 690-8504, Japan.
Shigeru
Kazutaka
K
Tsuchiya
Akiho
A
Itou
Noriko
N
Ohta
Yukari
Y
Tanaka
Katsunori
K
Nakagawa
Tsuyoshi
T
Matsuda
Hideyuki
H
Kawamukai
Makoto
M
D001426:Bacterial Proteins; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism; D017354:Point Mutation; D020560:Proteobacteria / Q000201:enzymology*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004220:Disulfides; D009844:Oligosaccharides; D018698:Glutamic Acid; C050523:chitohexaose; C418144:CSNA protein, Burkholderia gladioli; D006026:Glycoside Hydrolases; D003545:Cysteine