The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity.
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-alpha-L-arabinofuranosidase (MUAase) activity. A single MUAase gene (abf51A) was isolated, which encoded a non-modular glycoside hydrolase family (GH) 51 arabinofuranosidase (Abf51A) of 57000 Da. The substrate specificity of the Abf51A showed that it preferentially removed alpha1,2- and alpha1,3-linked arabinofuranose side chains from either arabinan or arabinoxylan, and hydrolysed alpha1,5-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.
Beylot
M H
MH
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK.
McKie
V A
VA
Voragen
A G
AG
Doeswijk-Voragen
C H
CH
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001089:Arabinose; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015723:Gene Library; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D009844:Oligosaccharides; D011994:Recombinant Proteins; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase