XynAF1 from Aspergillus fumigatus Z5 is an efficient thermophilic xylanase belonging to glycoside hydrolase family 10 (GH10). The non-catalytic amino acids N179 and R246 in its catalytic center formed one and three intermolecular H-bonds with the substrate in the aglycone region, respectively. Here we purified XynAF1-N179S and XynAF1-R246K, and obtained the protein-product complex structures by X-ray diffraction. The snapshots indicated that mutations at N179 and R246 had decreased the substrate-binding ability in the aglycone region. XynAF1-N179S, XynAF1-R246K, and XynAF1-N179S-R246K lost one, three, and four H-bonds with the substrate in comparison with the wild-type XynAF1, respectively, but this had little influence on the protein structure. As expected, N179S, R246K, and N179S-R246K led to a gradual decrease of substrate affinity of XynAF1. Interestingly, the enzyme assay showed that N179S increased catalytic efficiency, while both R246K and N179S-R246K had decreased catalytic efficiency. KEY POINTS: • The non-catalytic amino acids of XynAF1 could form H-bonds with the substrate. • The protein-product complex structures were obtained by X-ray diffraction. • The enzyme-substrate-binding capacity could affect enzyme catalytic efficiency.
Keywords: Enzyme catalysis; Enzyme-substrate binding; GH10 family xylanase.
© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.