Hydrocarbons are major constituents of petroleum, natural gas or essential oils and are primarily degraded by bacteria. The initial activation of apolar C‑H bonds of hydrocarbons represents a challenging task in chemical or enzymatic syntheses due to high activation energies. Under aerobic conditions, the hydroxylation of C-H bonds is catalysed by O2-dependent oxygenases or peroxidases, however, in the absence of oxygen, a different enzymatic strategy is required. In denitrifying bacteria, molybdenum-dependent hydroxylases of the type II DMSO reductase family were identified that use water for the hydroxylation of unactivated C-H bonds.

In this study, we have isolated and characterised the p‑cymene dehydrogenase (PCDH) from Aromatoleum aromaticum pCyN1¹ as a novel molybdenum-containing enzyme after heterologous production in the denitrifying host Thauera aromatica K172. Metal analyses and UV-vis spectroscopy of the heterotrimeric enzyme complex suggest the presence of a molybdenum cofactor, iron-sulfur clusters and a heme b. PCDH catalyses the hydroxylation of the primary carbon of the monoterpene p-cymene to its primary alcohol cuminol, and also the further oxidation to the corresponding aldehyde. Furthermore, we could show hydroxylation of a variety of aromatic hydrocarbons and terpenes to the corresponding primary alcohols, including p-xylene and limonene. This demonstrates the potential of this enzyme for the regioselective activation for a broad range of aromatic and isoprenoid compounds.

¹Strijkstra et al, AEM, 80(24), 7592–7603 (2014)