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

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 M-r, 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-beta 1,4-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-DNPG(2)) 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 dan 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-DNPG(2), E161A exhibited a much lower K-m, and k(cat) than native GalA, while E270A was inactive against the substrate. Analysis of the pre-steady-state kinetics of 2,4-DNPG(2) 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.