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

Single-domain auxiliary activity family 12 (AA12) pyrroloquinoline quinone-dependent dehydrogenases and free AA8 modules are prevalent in cellulolytic fungi; however, their function in polysaccharide biodegradation remains unclear. Here, we characterized three single-domain AA12 dehydrogenases (TthAA12, TthAA12B, and TteAA12A) and one free AA8 module (TthAA8A) from Thermothelomyces thermophilus and Thermothielavioides terrestris. All three single-domain AA12 dehydrogenases primarily function as dehydrogenases but also exhibit a measurable intrinsic oxidase activity, as evidenced by the concomitant production of H₂O₂. In addition to their known activity on several rare sugars, all three AA12 dehydrogenases showed weak activity toward xylo-oligosaccharides and D-xylulose. They directly transferred electrons to lytic polysaccharide monooxygenase (LPMO) and drove NcLPMO9C activity. The direct electron-transfer capability and unique substrate specificities of the three single-domain AA12 dehydrogenases stand in marked contrast to the AA8 cytochrome domain-dependent mechanism of the previously characterized multi-domain CcPDH. The free TthAA8A module exhibited weak electron-accepting ability from AA12 dehydrogenases, yet it effectively donated electrons to NcLPMO9C or cytochrome c. In addition, it attenuated H₂O₂ production, thereby boosting the catalytic efficiency of AA12 dehydrogenase-driven NcLPMO9C. Taken together, these findings expand the known repertoire of auxiliary redox partners that support LPMO activity in hemi-/cellulolytic fungal enzyme cocktails.