Biochem. J. (2006) 394
(675–686) (Printed in Great Britain)
Two exo-b-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases
Nathalie CÔTÉ*1, Alain FLEURY*,Émilie DUMONT-BLANCHETTE*, Tamo FUKAMIZO†, Masaru MITSUTOMI‡ and Ryszard BRZEZINSKI*2
*Centre d'Étude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1, †Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan, and ‡Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan
A GlcNase (exo-b-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 Km 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 b-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 b-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 b-galactosidase, b-glucuronidase or b-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.
Key words: amino sugar, chitosan hydrolysis, csxA gene, exo-glucosaminidase, glycoside hydrolase, Streptomyces.
Abbreviations used: CBM, carbohydrate-binding module; GH, glycoside hydrolase family; GlcN, D-glucosamine; GlcNAc, N-acetylglucosamine; GlcNase, exo-b-D-glucosaminidase; ORF, open reading frame; p-NP, p-nitrophenyl; SP-Sepharose, sulphopropyl-Sepharose; YME medium, yeast/malt extract medium.
1Present address: ISM Biopolymer, 220 rue Denison Est, Granby, QC, Canada, J2H 2R6.
2To whom correspondence should be addressed (email Ryszard.Brzezinski@USherbrooke.ca).
The nucleotide sequence reported in this paper has been submitted to the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AY962188.
Received 30 August 2005/28 November 2005; accepted 29 November 2005
Published as BJ Immediate Publication 29 November 2005, doi:10.1042/BJ20051436
The Biochemical Society, London ©2006
Editorial Advisory Panel
RSS feeds
Table of contents by email
My BJ toolbox
