A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.
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.
Ueda
Mitsuhiro
M
Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Japan.
Kojima
Miki
M
Yoshikawa
Tomoye
T
Mitsuda
Norio
N
Araki
Keiichi
K
Kawaguchi
Takashi
T
Miyatake
Kazutaka
K
Arai
Motoo
M
Fukamizo
Tamo
T
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
D006027:Glycosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C041162:N-acetylglucosamine hexasaccharide 1-4; D004247:DNA; D002688:Chitinases