The CBM91 module enhances the activity of β-xylosidase/α-L-arabinofuranosidase PphXyl43B from Paenibacillus physcomitrellae XB by adopting a unique loop conformation at the top of the active pocket

Abstract

Carbohydrate-binding module (CBM) family 91 is a novel module primarily associated with glycoside hydrolase (GH) family 43 enzymes. However, our current understanding of its function remains limited. PphXyl43B is a β-xylosidase/α-L-arabinofuranosidase bifunctional enzyme from physcomitrellae patens XB belonging to the GH43_11 subfamily and containing CBM91 at its C terminus. To fully elucidate the contributions of the CBM91 module, the truncated proteins consisting only the GH43_11 catalytic module (rPphXyl43B-dCBM91) and only the CBM91 module (rCBM91) of PphXyl43B were constructed, respectively. The result showed that rPphXyl43B-dCBM91 completely lost hydrolysis activity against both p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside; it also exhibited significantly reduced activity towards xylobiose, xylotriose, oat spelt xylan and corncob xylan compared to the control. Thus, the CBM91 module is crucial for the β-xylosidase/α-L-arabinofuranosidase activities in PphXyl43B. However, rCBM91 did not exhibit any binding capability towards corncob xylan. Structural analysis indicated that CBM91 of PphXyl43B might adopt a loop conformation (residues 496–511: ILSDDYVVQSYGGFFT) to actively contribute to the catalytic pocket formation rather than substrate binding capability. This study provides important insights into understanding the function of CBM91 and can be used as a reference for analyzing the action mechanism of GH43_11 enzymes and their application in biomass energy conversion.