Structure-based mutagenesis of Penicillium griseofulvum xylanase using computational design.

International audiencePenicillium griseofulvum xylanase (PgXynA) belongs to family 11 glycoside hydrolase. It exhibits unique amino acid features but its three-dimensional structure is not known. Based upon the X-ray structure of Penicillium funiculosum xylanase (PfXynC), we generated a three-dimensional model of PgXynA by homology modeling. The native structure of PgXynA displayed the overall beta-jelly roll folding common to family 11 xylanases with two large beta-pleated sheets and a single alpha-helix that form a structure resembling a partially closed right hand. Although many features of PgXynA were very similar to previously described enzymes from this family, crucial differences were observed in the loop forming the "thumb" and at the edge of the binding cleft. The robustness of the xylanase was challenged by extensive in silico-based mutagenesis analysis targeting mutations retaining stereochemical and energetical control of the protein folding. On the basis of structural alignments, modeled three-dimensional structure, in silico mutations and docking analysis, we targeted several positions for the replacement of amino acids by site-directed mutagenesis to change substrate and inhibitor specificity, alter pH profile and improve overall catalytic activity. We demonstrated the crucial role played by Ser44(PgXynA) and Ser129(PgXynA), two residues unique to PgXynA, in conferring distinct specificity to P. griseofulvum xylanase. We showed that the pH optimum of PgXynA could be shifted by -1 to +0.5 units by mutating Ser44(PgXynA) to Asp and Asn, respectively. The S44D and S44N mutants showed only slight alteration in K(m) and V(max) whereas a S44A mutant lost both pH-dependence profile and activity. We were able to produce PgXynA S129G mutants with acquired sensitivity to the Xylanase Inhibitor Protein, XIP-I. The replacement of Gln121(PgXynA), located at the start of the thumb, into an Arg residue resulted in an enzyme that possessed a higher catalytic activity

Use of family 8 enzymes with xylanolytic activity in baking

The present invention describes a method to improve the properties of a dough and/or a baked product by adding a bread or dough-improving agent containing a enzyme with xylanolytic activity belonging to glycoside hydrolases family 8. Preferred enzymes are the psychrophilic xylanase from Pseudoalteromonas haloplanktis and the mesophilic xylanase Y from Bacillus halodurans C-125

Use of family 8 enzymes with xylanolytic activity in baking

The present invention describes a method to improve the properties of a dough and/or a baked product by adding a bread or dough-improving agent containing a enzyme with xylanolytic activity belonging to glycoside hydrolases family 8. Preferred enzymes are the psychrophilic xylanase from Pseudoalteromonas haloplanktis and the mesophilic xylanase Y from Bacillus halodurans C-125

Molecular determinants of substrate and inhibitor specificities of the Penicillium griseofulvum family 11 xylanases.

Penicillium griseofulvum possesses two endo-(1,4)-beta-xylanase genes, PgXynA and PgXynB, belonging to family 11 glycoside hydrolases. The enzymes share 69% identity, a similar hydrolysis profile i.e. the predominant production of xylobiose and xylotriose as end products from wheat arabinoxylan and a specificity region of six potential xylose subsites, but differ in terms of catalytic efficiency which can be explained by subtle structural differences in the positioning of xylohexaose in the PgXynB model. Site-directed mutagenesis of the "thumb" region revealed structural basis of PgXynB substrate and inhibitor specificities. We produced variants displaying increased catalytic efficiency towards wheat arabinoxylan and xylo-oligosaccharides and identified specific determinants in PgXynB "thumb" region responsible for resistance to the wheat xylanase inhibitor XIP-I. Based on kinetic analysis and homology modeling, we suggested that Pro130(PgXynB), Lys131(PgXynB) and Lys132(PgXynB) hamper flexibility of the loop forming the "thumb" and interfere by steric hindrance with the inhibitor