Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans

Abstract

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.

Key Residues in Subsite F Play a Critical Role in the Activity of Pseudomonas fluorescens Subspecies cellulosa Xylanase A Against Xylooligosaccharides but Not Against Highly Polymeric Substrates such as Xylan

“…It is possible that xylan forms such strong interactions with other residues within the active site cleft that removal of the C-2-O/E43 interaction does not alter the position of the substrate within the enzyme. This hypothesis is in agreement with Moreau et al (29), who also demonstrated that a mutation within XYLASL, D124E, had a much greater effect on the affinity of the enzyme for PNPC compared with xylan.…”

“…It remains to be established whether Cex or XYLA represents the best paradigm for family 10 xylanases. However, the observation that XYLASL has a K m for PNPC intermediate between XYLA and Cex (29) suggests that family 10 enzymes will exhibit a range of different activities for the aryl ␤-glycosides.…”

A Xylanase, AtXyn1, is Predominantly Expressed in Vascular Bundles, and Four Putative Xylanase Genes were Identified in the Arabidopsis thaliana Genome

“…Replacement of E-128 or E-236 by isosteric glutamine completely abolished its enzymatic activity (Moreau et al 1994). Furthermore, Henrissat identified the sequence around E-236 of XlnA 32kD as the glycosyl hydrolase family-10 active site (Henrissat 1997).…”

X-ray crystallographic study of xylopentaose binding toPseudomonas fluorescens xylanase A

“…Mutagenesis of the equivalent carboxylates also showed loss of catalytic activity in Streptomyces lividans xylanase A 21 (XAS) and XYLA. 4,22 Family 10 belongs in turn to the 4/7 superfamily 23 (or clan GH-A 24 ) of TIM-barrels, which includes glycosyl hydrolases with diverse specificities but a common structural framework, conserved catalytic residues, and common evolutionary origin.…”