Mannanase A from Pseudomonas fluorescens ssp. cellulosa is a retaining glycosyl hydrolase in which E212 and E320 are the putative catalytic residues

Biochemistry
D N BolamHarry J Gilbert

Abstract

Mannanase A (MANA) from Pseudomonas fluorescens, a member of glycosyl hydrolase family 26, was hyperexpressed in Escherichia coli and purified to homogeneity. Analysis of the stereochemical course of mannotetraose hydrolysis by purified MANA showed that the configuration of the anomeric carbon was retained on cleavage of the middle glycosidic bond. These data suggest that the mannanase hydrolyzes mannooligosaccharides by a double-displacement general acid-base mechanism. By hydrophobic cluster analysis (HCA), two glutamate and two aspartate residues were shown to be conserved in all of the glycosyl hydrolase family 26 enzymes analyzed. In addition, HCA suggested that family 26 was related to the GH-A clan (families 1, 2, 5, 10, 30, 35, 39, and 42) of (alpha/beta)8-barrel glycosyl hydrolases, which led to the prediction that E320 and E212 constitute the catalytic nucleophile and acid-base residues, respectively. To investigate the role of these amino acids, site-directed mutagenesis was used to replace the two aspartates with alanine and glutamate, while the two conserved glutamates were changed to alanine and aspartate. The mutant enzymes were purified and their biochemical properties were analyzed. The data showed that neither...Continue Reading

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Citations

Nov 15, 2006·Applied Microbiology and Biotechnology·Nisha PalackalBrian A Steer
Feb 3, 2012·Journal of the American Chemical Society·Ethan D Goddard-BorgerStephen G Withers
Dec 21, 2012·Journal of Agricultural and Food Chemistry·Priti KatroliaZhengqiang Jiang
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