Two exo-beta-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases.
A GlcNase (exo-beta-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with K(m) and kcat values of 0.16 mg/ml and 2832 min(-1). On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative beta-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable beta-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with beta-galactosidase, beta-glucuronidase or beta-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.
Côté
Nathalie
N
Centre d'Etude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1.
Fleury
Alain
A
Dumont-Blanchette
Emilie
E
Fukamizo
Tamo
T
Mitsutomi
Masaru
M
Brzezinski
Ryszard
R
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D013379:Substrate Specificity
D006026:Glycoside Hydrolases