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
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- 作者：Kerynne L. Braithwaite ; Tereza Barna ; Tracey D. Spurway ; Simon J. Charnock ; Gary W. Black ; Neil Hughes ; Jeremy H. Lakey ; Richard Virden ; Geoffrey P. Hazlewood ; Bernard Henrissat ; and Harry J. Gilbert
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：December 9, 1997
- 年：1997
- 卷：36
- 期：49
- 页码：15489 - 15500
- 全文大小：290K
- 年卷期：v.36,no.49(December 9, 1997)
- ISSN：1520-4995
文摘
A genomic library of Pseudomonas fluorescenssubsp. cellulosa DNA was screened forgalactanase-positive recombinants. The nine galactanase positivephage isolated contained the samegalactanase gene designated galA. The deduced primarystructure of the enzyme (galactanase A; GalA)encoded by galA had a Mr of 42 130and exhibited significant sequence identity with a galactanasefromAspergillus aculeatus, placing GalA in glycosyl hydrolasefamily 53. The enzyme displayed propertiestypical of an endo-
1,4-galactanase and exhibited no activity againstthe other plant structuralpolysaccharides evaluated. Analysis of the stereochemical courseof 2,4-dinitrophenyl--galactobioside(2,4-DNPG2) hydrolysis by GalA indicated that thegalactanase catalyzes the hydrolysis of glycosidicbonds by a double displacement general acid-base mechanism.Hydrophobic cluster analysis (HCA)suggested that family 53 enzymes are related to the GH-A clan ofglycosyl hydrolases, which have an(
/)8 barrel structure. HCA also predicted thatE161 and E270 were the acid-base and nucleophilicresidues, respectively. Mutants of GalA in which E161 and E270 hadbeen replaced with alanine residueswere essentially inactive against galactan. Against2,4-DNPG2, E161A exhibited a much lowerKm andkcat than native GalA, while E270A was inactiveagainst the substrate. Analysis of thepre-steady-statekinetics of 2,4-DNPG2 hydrolysis by E161A showed that therewas an initial rapid release of2,4-dinitrophenol (2,4-DNP), which then decayed to a slow steady-staterate of product formation. Nopre-steady-state burst of 2,4-DNP release was observed with thewild-type enzyme. These data areconsistent with the HCA prediction that E161 and E270 are theacid-base and nucleophilic catalyticresidues of GalA, respectively.
1,4-galactanase and exhibited no activity againstthe other plant structuralpolysaccharides evaluated. Analysis of the stereochemical courseof 2,4-dinitrophenyl--galactobioside(2,4-DNPG2) hydrolysis by GalA indicated that thegalactanase catalyzes the hydrolysis of glycosidicbonds by a double displacement general acid-base mechanism.Hydrophobic cluster analysis (HCA)suggested that family 53 enzymes are related to the GH-A clan ofglycosyl hydrolases, which have an(/)8 barrel structure. HCA also predicted thatE161 and E270 were the acid-base and nucleophilicresidues, respectively. Mutants of GalA in which E161 and E270 hadbeen replaced with alanine residueswere essentially inactive against galactan. Against2,4-DNPG2, E161A exhibited a much lowerKm andkcat than native GalA, while E270A was inactiveagainst the substrate. Analysis of thepre-steady-statekinetics of 2,4-DNPG2 hydrolysis by E161A showed that therewas an initial rapid release of2,4-dinitrophenol (2,4-DNP), which then decayed to a slow steady-staterate of product formation. Nopre-steady-state burst of 2,4-DNP release was observed with thewild-type enzyme. These data areconsistent with the HCA prediction that E161 and E270 are theacid-base and nucleophilic catalyticresidues of GalA, respectively.