Biochem. J. (2001) 358
(607–614) (Printed in Great Britain)
The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity
Marie-Helene BEYLOT*, Vincent A. MCKIE*, Alphons G. J. VORAGEN†, Chantal H. L. DOESWIJK-VORAGEN† and Harry J. GILBERT*1
*Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K, and †Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands
To investigate the mechanism by which Pseudomonas cellulosa releases arabinose from polysaccharides and oligosaccharides, a gene library of P. cellulosa genomic DNA was screened for 4-methylumbelliferyl-a-L-arabinofuranosidase (MUAase) activity. A single MUAase gene (abf51A) was isolated, which encoded a non-modular glycoside hydrolase family (GH) 51 arabinofuranosidase (Abf51A) of 57000Da. The substrate specificity of the Abf51A showed that it preferentially removed a1,2- and a1,3-linked arabinofuranose side chains from either arabinan or arabinoxylan, and hydrolysed a1,5-linked arabino-oligosaccharides, although at a much lower rate. The activity of Abf51A against arabinoxylan was similar to a GH62 arabinofuranosidase encoded by a P. cellulosa gene. Glu-194 and Glu-321 of Abf51A are conserved in GH51 enzymes, and it has been suggested that these amino acids comprise the key catalytic acid/base and nucleophile residues, respectively. To evaluate this hypothesis the biochemical properties of E194A and E321A mutants of Abf51A were evaluated. The data were consistent with the view that Glu-194 and Glu-321 comprise the key catalytic residues of Abf51A. These data, in conjunction with the results presented in the accompanying paper [Beylot, Emami, McKie, Gilbert and Pell (2001) Biochem. J. 358, 599–605], indicate that P. cellulosa expresses a membrane-bound GH51 arabinofuranosidase that plays a pivotal role in releasing arabinose from a range of polysaccharides and oligosaccharides.
Key words: arabinose, catalytic mutant.
Abbreviations used: GH, glycoside hydrolase family; MUA, 4-methylumbelliferyl-a-L-arabinofuranoside; MUAase, 4-methylumbelliferyl-a-L-arabinofuranosidase; LB, Luria–Bertani broth; IPTG, isopropyl b-D-thiogalactoside; HPAEC, high-performance anion-exchange chromatography; ORF, open reading frame.
1To whom correspondence should be addressed (e-mail H. J.Gilbert@Newcastle.ac.uk).
The nucleotide sequence data reported for abf51A and flanking sequences will appear in the GenBank® Nucleotide Sequence Databases under the accession number AY043167.
Received 23 February 2001/30 May 2001; accepted 6 July 2001
The Biochemical Society, London ©
2001
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