Oxyfunctionalization of Primary Benzylic C(sp3)–H Bonds with Water by a Molybdenum-Dependent Dehydrogenase

The selective oxyfunctionalization of C(sp3)–H bonds remains a central challenge in chemical synthesis. Although oxygenases and peroxygenases provide biocatalytic alternatives to metal-based chemical methods, their practical use is constrained by costly cofactor regeneration, toxic co-substrates, or limited selectivity. Molybdenum (Mo)-dependent alkyl dehydrogenases circumvent these limitations by employing water, rather than O2 or H2O2, as the oxygen source for hydroxylation in conjunction with an electron acceptor. Here, we combine enzymatic, structural, and computational analyses to characterize a Mo-dependent p-cymene dehydrogenase (pCDH) from an anaerobic hydrocarbon-degrading bacterium. Isolated pCDH selectively hydroxylates a variety of benzylic and allylic C(sp3)–H bonds. A 2.0 Å cryo-EM structure of the heterotrimeric enzyme in complex with p-cymene captures key states of the catalytic cycle. Structure-guided QM/MM modelling and docking rationalize the substrate scope and radical mechanism. These results identify water-dependent Mo-dehydrogenases as promising catalysts for selective C(sp3)–H oxyfunctionalization.