Jacques Georis

Substrate and product hydrolysis specificity in family 11 glycoside hydrolases : an analysis of Penicillium funiculosum and Penicillium griseofulvum xylanases

Two genes encoding family 11 endo-(1,4)-β-xylanases from Penicillium griseofulvum (PgXynA) and Penicillium funiculosum (PfXynC) were heterologously expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the recombinant enzymes were purified after affinity chromatography and proteolysis. PgXynA and PfXynC were identical to their native counterparts in terms of molecular mass, pI, N-terminal sequence, optimum pH, and enzymatic activity towards arabinoxylan. Further investigation of the rate and pattern of hydrolysis of PgXynA and PfXynC on wheat soluble arabinoxylan showed the predominant production of xylotriose and xylobiose as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing chain length (n) of oligomer up to n = 6, suggesting that the specificity region of both Penicillium xylanases spans about six xylose units. In contrast to PfXynC, PgXynA was found insensitive to the wheat xylanase inhibitor protein XIP-I.

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

Penicillium 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 β‐jelly roll folding common to family 11 xylanases with two large β‐pleated sheets and a single α‐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 Ser44PgXynA and Ser129PgXynA, 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 Ser44PgXynA to Asp and Asn, respectively. The S44D and S44N mutants showed only slight alteration in Km and Vmax 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 Gln121PgXynA, located at the start of the thumb, into an Arg residue resulted in an enzyme that possessed a higher catalytic activity. Proteins 2008.