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Biochemistry. 1997 Dec 9;36(49):15489-500.

Evidence that galactanase A from Pseudomonas fluorescens subspecies cellulosa is a retaining family 53 glycosyl hydrolase in which E161 and E270 are the catalytic residues.

Author information

1
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.

Abstract

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was screened for galactanase-positive recombinants. The nine galactanase positive phage isolated contained the same galactanase gene designated galA. The deduced primary structure of the enzyme (galactanase A; GalA) encoded by galA had a Mr of 42 130 and exhibited significant sequence identity with a galactanase from Aspergillus aculeatus, placing GalA in glycosyl hydrolase family 53. The enzyme displayed properties typical of an endo-beta1, 4-galactanase and exhibited no activity against the other plant structural polysaccharides evaluated. Analysis of the stereochemical course of 2,4-dinitrophenyl-beta-galactobioside (2,4-DNPG2) hydrolysis by GalA indicated that the galactanase catalyzes the hydrolysis of glycosidic bonds by a double displacement general acid-base mechanism. Hydrophobic cluster analysis (HCA) suggested that family 53 enzymes are related to the GH-A clan of glycosyl hydrolases, which have an (alpha/beta)8 barrel structure. HCA also predicted that E161 and E270 were the acid-base and nucleophilic residues, respectively. Mutants of GalA in which E161 and E270 had been replaced with alanine residues were essentially inactive against galactan. Against 2,4-DNPG2, E161A exhibited a much lower Km and kcat than native GalA, while E270A was inactive against the substrate. Analysis of the pre-steady-state kinetics of 2,4-DNPG2 hydrolysis by E161A showed that there was an initial rapid release of 2,4-dinitrophenol (2,4-DNP), which then decayed to a slow steady-state rate of product formation. No pre-steady-state burst of 2,4-DNP release was observed with the wild-type enzyme. These data are consistent with the HCA prediction that E161 and E270 are the acid-base and nucleophilic catalytic residues of GalA, respectively.

PMID:
9398278
DOI:
10.1021/bi9712394
[Indexed for MEDLINE]

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