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 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.
Braithwaite
K L
KL
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
Barna
T
T
Spurway
T D
TD
Charnock
S J
SJ
Black
G W
GW
Hughes
N
N
Lakey
J H
JH
Virden
R
R
Hazlewood
G P
GP
Henrissat
B
B
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002384:Catalysis; D004274:DNA, Recombinant; D005685:Galactans / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D005685:Galactans; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C109490:galactanase A