Discovery of a Novel Cellulase ZF580 From Mount Everest Metagenome Featuring a Catalytically Active DUF5916 Domain

Cellulases are crucial biocatalysts with extensive industrial applications, yet their study has been constrained by cultivation limitations of native microorganisms. Here, we report the discovery and characterization of a novel multifunctional cellulase (ZF580) from the extreme environment of Mount Everest using metagenomic approaches. Functional screening revealed ZF580's unique capacity to hydrolyze diverse substrates, including 4-nitrophenyl-β-D-glucopyranoside (pNPG), chitin, microcrystalline cellulose, and carboxymethyl cellulose sodium (CMC-Na). Phylogenetically, ZF580 forms an independent clade distinct from characterized β-glucosidases and known glycoside hydrolase (GH) families, suggesting its classification as a progenitor of a novel GH lineage. Structural modeling revealed a distinctive (β/α)8 TIM-barrel fold, diverging from canonical GH family architectures. Crucially, truncation analysis and site-directed mutagenesis identified the previously uncharacterized Domain of Unknown Function 5916 (DUF5916) as a catalytic functional region, with residue E373 serving as its essential proton donor. This study provides the first experimental evidence of DUF5916's enzymatic activity, redefining it as a novel catalytic domain. Overall, these findings suggest that ZF580 is a cellulolytic enzyme with β-glucosidase activity and that DUF5916 forms its catalytic core, offering insights that may be valuable for future studies on enzyme function and engineering.