title: A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.
abstract: A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.
authors.affiliation: Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Ueda
authors.forename: Miki
authors.initials: M
authors.lastname: Kojima
authors.forename: Tomoye
authors.initials: T
authors.lastname: Yoshikawa
authors.forename: Norio
authors.initials: N
authors.lastname: Mitsuda
authors.forename: Keiichi
authors.initials: K
authors.lastname: Araki
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawaguchi
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Miyatake
authors.forename: Motoo
authors.initials: M
authors.lastname: Arai
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D000333:Aeromonas / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D006027:Glycosides / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D006027:Glycosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C041162:N-acetylglucosamine hexasaccharide 1-4; D004247:DNA; D002688:Chitinases