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bioRxiv · 10.1101/2025.09.02.673750

Novel single-domain AA12 pyrroloquinoline quinone-dependent oxidoreductases directly and co-operatively drive lytic polysaccharide monooxygenase activity with AA8 module

Sun, Y.·Yuan, C.·Long, L.·Ding, S.
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Abstract

The single-domain auxiliary activity family 12 (AA12) pyrroloquinoline quinone-dependent oxidoreductases and free AA8 modules are prevalent in cellulolytic fungi, however, their function in polysaccharide biodegradation is still confused. Here, we characterized three single-domain AA12 oxidoreductases and one free AA8 module from Thermothelomyces thermophilus and Thermothielavioides terrestris. All three single-domain AA12 oxidoreductases are restrict dehydrogenases with trace oxidase activity. All three single-domain AA12 enzymes could directly transfer electrons to lytic polysaccharide monooxygenase (LPMO) and drive NcLPMO9C activity. Furthermore, inter-protein electron transfer between single-domain AA12 enzymes and the AA8 module was observed. The AA12 enzyme-driven NcLPMO9C efficiency could be significantly enhanced by the addition of free AA8 module TthAA8B, probably attributing to the acceleration of electron transfer from AA12 enzymes to NcLPMO9C and the attenuation of H2O2 accumulation mediated by TthAA8B. Our findings highlight the potential role of single-domain AA12 enzyme and free AA8 modules in the biodegradation system of LPMOs. IMPORTANCEThis study reveals the directly electron transferring and driving capability of single-domain AA12 PQQ-dependent enzyme for the oxidative reaction of lytic polysaccharide monooxygenase (LPMO). This funding is quite distinct from the AA8 cytochrome domain-dependent driving pattern of the previous characterized multi-domain CcPDH. We also demonstrated that this priming capability could be facilitated by the free AA8 cytochrome module, providing new insight on the interactions and functions of single-domain AA12 enzymes and free AA8 modules in fueling LPMO activity during fungal lignocellulose biodegradation process. These findings collectively provide evidence for the potential function of widespread single-domain AA12 PQQ-dependent enzymes and free AA8 cytochrome modules as unique enzyme redox partners in cellulolytic fungi.

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Sun, Y., Yuan, C., Long, L., Ding, S.. 2025-09-03. Novel single-domain AA12 pyrroloquinoline quinone-dependent oxidoreductases directly and co-operatively drive lytic polysaccharide monooxygenase activity with AA8 module. https://doi.org/10.1101/2025.09.02.673750

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