A unique loop conformation confers the enhanced catalytic efficiency of the GH10 endoxylanase catalytic domain Xyn10B...

GH10 endo-β-1,4-xylanases are widely distributed enzymes that play a pivotal role in the degradation of heterogeneous xylans. A comprehensive understanding of catalytic efficiency and underlying mechanisms is essential for their effective application. In this study, the catalytic domains Xyn10A and Xyn10B from two GH10_e130 enzymes of Paenibacillus physcomitrellae XB were comparatively characterized. Xyn10B exhibited significantly higher activity toward xylans and xylooligosaccharides, predominantly producing xylobiose; whereas Xyn10A displayed lower activity and generated both xylobiose and xylotriose as major products. Structural analyses revealed a unique loop conformation (FWINSKPDVN, residues 246-255) situated between the β6 strand and the α6 helix-exclusively present in Xyn10B. Deletion mutagenesis targeting the KPDVN region demonstrated that progressive removal of residues substantially reduced hydrolytic efficiency. Notably, deletion of DVN shifted the hydrolysis profile to resemble that of Xyn10A; deletions of PDVN or KPDVN resulted in nearly complete loss of activity toward xylotriose. Further alanine-scanning mutagenesis of five key residues indicated that this loop might form a sliding guide clamp via the structural rigidity conferred by P252 to enhance the processive cleavage of long-chain substrates and also act as a substrate concentration-dependent conformational filter to selectively promote hydrolytic catalysis of xylotriose. As the key structural determinant underlying the functional divergence between Xyn10B and Xyn10A, this loop underpins enhanced catalytic efficiency and hydrolytic processivity. These findings provide mechanistic insights into GH10 endoxylanases and establish a structural framework for rational engineering aimed at improving catalytic performance and tailoring product profiles.