Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain.
Cells of "Paenibacillus fukuinensis" D2 produced chitosanase into surrounding medium, in the presence of colloidal chitosan or glucosamine. The gene of this enzyme was cloned, sequenced, and subjected to site-directed mutation and deletion analyses. The nucleotide sequence indicated that the chitosanase was composed of 797 amino acids and its molecular weight was 85,610. Unlike conventional family 46 chitosanases, the enzyme has family 8 glycosyl hydrolase catalytic domain, at the amino-terminal side, and discoidin domain at the carboxyl-terminal region. Expression of the cloned gene in Escherichia coli revealed beta-1,4-glucanase function, besides chitosanase activity. Analyses by zymography and immunoblotting suggested that the active enzyme was, after removal of signal peptide, produced from inactive 81-kDa form by proteolysis at the carboxyl-terminal region. Replacements of Glu(115) and Asp(176), highly conserved residues in the family 8 glycosylase region, with Gln and Asn caused simultaneous loss of chitosanase and glucanase activities, suggesting that these residues formed part of the catalytic site. Truncation experiments demonstrated indispensability of an amino-terminal region spanning 425 residues adjacent to the signal peptide.
Kimoto
Hisashi
H
Department of Biochemistry I, Faculty of Medicine, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan.
Kusaoke
Hideo
H
Yamamoto
Ikkyu
I
Fujii
Yutaka
Y
Onodera
Takashi
T
Taketo
Akira
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D000070821:Discoidins; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037102:Lectins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D000070821:Discoidins; D037102:Lectins; D015800:Protozoan Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase