EVIDENCE THAT GALACTANASE A FROM PSEUDOMONAS-FLUORESCENS SUBSPECIES CELLULOSA IS A RETAINING FAMILY-53 GLYCOSYL HYDROLASE IN WHICH E161 ANDE270 ARE THE CATALYTIC RESIDUES

A genomic library of Pseudomonas fluorescens subsp, cellulosa DNA was screened for galactanase-positive recombinants. The nine galactanase p ositive 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 seque nce identity with a galactanase from Aspergillus aculeatus, placing Ga lA in glycosyl hydrolase family 53. The enzyme displayed properties ty pical of an endo-beta 1,4-galactanase and exhibited no activity agains t the other plant structural polysaccharides evaluated, Analysis of th e stereochemical course of 2,4-dinitrophenyl-beta-galactobioside (2,4- DNPG(2)) hydrolysis by GalA indicated that the galactanase catalyzes t he hydrolysis of glycosidic bonds by a double displacement general aci d-base mechanism, :Hydrophobic cluster analysis (HCA) suggested that f amily 53 enzymes are related to the GH-A dan of glycosyl hydrolases, w hich have an (alpha/beta)(8) barrel structure, HCA also predicted that E161 and E270 were the acid-base and nucleophilic residues, respectiv ely, Mutants of GalA in which E161 and E270 had been replaced with ala nine 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 t here was an initial rapid release of 2,4-dinitrophenol (2,4-DNP), whic h then decayed to a slow steady-state rate of product formation. No pr e-steady-state burst of 2,4-DNP release was observed with the wild-typ e enzyme, These data are consistent with the HCA prediction that E161 and E270 are the acid-base and nucleophilic catalytic residues of GalA , respectively.