Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action.
Pseudomonas fluorescens subsp. cellulosa expressed arabinanase activity when grown on media supplemented with arabinan or arabinose. Arabinanase activity was not induced by the inclusion of other plant structural polysaccharides, and was repressed by the addition of glucose. The majority of the Pseudomonas arabinanase activity was extracellular. Screening of a genomic library of P. fluorescens subsp. cellulosa DNA constructed in Lambda ZAPII, for recombinants that hydrolysed Red-dyed arabinan, identified five arabinan-degrading plaques. Each of the phage contained the same Pseudomonas arabinanase gene, designated arbA, which was present as a single copy in the Pseudomonas genome. The nucleotide sequence of arbA revealed an open reading frame of 1041 bp encoding a protein, designated arabinanase A (ArbA), of Mr 39438. The N-terminal sequence of ArbA exhibited features typical of a prokaryotic signal peptide. Analysis of the primary structure of ArbA indicated that, unlike most Pseudomonas plant cell wall hydrolases, it did not contain linker sequences or have a modular structure, but consisted of a single catalytic domain. Sequence comparison between the Pseudomonas arabinanase and proteins in the SWISS-PROT database showed that ArbA exhibits greatest sequence identity with arabinanase A from Aspergillus niger, placing the enzyme in glycosyl hydrolase Family 43. The significance of the differing substrate specificities of enzymes in Family 43 is discussed. ArbA purifed from a recombinant strain of Escherichia coli had an Mr of 34000 and an N-terminal sequence identical to residues 32-51 of the deduced sequence of ArbA, and hydrolysed linear arabinan, carboxymethylarabinan and arabino-oligosaccharides. The enzyme displayed no activity against other plant structural polysaccharides, including branched sugar beet arabinan. ArbA produced almost exclusively arabinotriose from linear arabinan and appeared to hydrolyse arabino-oligosaccharides by successively releasing arabinotriose. ArbA and the Aspergillus arabinanase mediated a decrease in the viscosity of linear arabinan that was associated with a significant release of reducing sugar. We propose that ArbA is an arabinanase that exhibits both an endo- and an exo- mode of action.
McKie
V A
VA
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, UK.
Black
G W
GW
Millward-Sadler
S J
SJ
Hazlewood
G P
GP
Laurie
J I
JI
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D015183:Restriction Mapping
D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; C082526:arabinan endo-1,5-alpha-L-arabinosidase