Mannanase A from Pseudomonas fluorescens ssp. cellulosa Is a Retaining Glycosyl Hydrolase in Which E212 and E320 Are the Putative Catalytic Residues

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 the D-->A nor the D-->E mutation resulted in a dramatic decrease in enzyme activity, suggesting that the two aspartate residues did not play a pivotal role in catalysis. In contrast, modification of either of the glutamate residues to alanine caused a dramatic decrease in kcat against carob galactomannan, azo-carob galactomannan, mannotetraose and 2,4-dinitrophenyl beta-mannobioside (2,4-DNPM). The E320A mutation did not alter the apparent K(m) (K(m)) of MANA against these substrates, while E212A resulted in a 27-fold decrease in K(m) against 2,4-DNPM. Pre-steady-state kinetics of 2,4-DNPM hydrolysis by E212A showed that there was a rapid burst of 2,4-dinitrophenol release. Circular dichroism and fluorescence spectroscopy indicated that there were no significant differences between the structures of the mutant and wild-type forms of MANA. These data are consistent with E212 and E320 constituting the catalytic acid-base and nucleophile residues of MANA, respectively.

A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose

“…For example, some of the pectinases may not have attacked the dyed substrates, the pectinase genes are not expressed in E. coli, or the polysaccharidase genes are not represented in the gene library, as they are lethal to the enteric bacterium. This last possibility is consistent with the observation that the high-level expression of several P. cellulosa glycoside hydrolases is lethal to E. coli, due to inefficient secretion [17,30,31].…”

A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi The GenBank/EMBL accession number for the sequence reported in this paper is AJ242666.

“…1). Bolam et al (1996) demonstrated that two glutamate residues act as the catalytic nucleophile and acid-base residues. It is widely believed that these residues are highly conserved within members of the same family and sequence homology suggested that E339 and E438 act as the acid-base and nucleophilic residues, respectively, in C. thermocellum Man26A.…”

The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism

“…The resultant DNA was then cloned into the BamHI site of pJG5h, and recombinants in which the two regions of xynA were in the same orientation were designated pJG5, and retained for further use. The conditions used in all the PCR reactions were as described previously [25]. The plasmid pJG4 was constructed by subjecting pJG2 to site-directed mutagenesis using the Transformer kit supplied by ClonTech (Heidelberg, Germany).…”