Hydrocarbons are essential in numerous natural processes and represent the main components of crude oil.1 The conversion of hydrocarbons into downstream products represents the basis for the petrochemical, pharmaceutical and plastic industries. Made up primarily of hydrogen and carbon atoms, hydrocarbons exist in various forms, including alkanes, alkenes and aromatic compounds. Their degradation is essential for the bioremediation of polluted environments, as they can be toxic and persistent in nature1. On the other side, the initial steps of bacterial alkane degradation may be important for the functionalization of alkanes into chiral alcohols as building blocks for value-added products. The activation of non-polar C‑H bonds in hydrocarbons is a major challenge in both chemical and enzymatic syntheses due to high activation energies. In aerobic environments, bacteria use O2 for the hydroxylation of C-H bonds catalysed by oxygenases or peroxygenases. In contrast, anaerobic bacteria employ alternative strategies for the initial hydrocarbon activation. Here, molybdenum-dependent hydroxylases of the type II DMSO reductase family have been postulated as key enzymes that use water to hydroxylate unactivated C-H bonds.
In this study, we isolated and characterized a p‑cymene dehydrogenase (CmdABC) from denitrifying Aromatoleum aromaticum pCyN12 as well as a putative alkane hydroxylase (AhyABC) from the sulfate-reducing bacterium Desulfococcus oleovorans Hxd33,4 after heterologous production in the β‑proteobacterium Thauera aromatica K172. Metal analyses and UV-vis spectroscopy of the heterotrimeric enzyme complexes indicate the presence of one molybdenum cofactor, five FeS clusters and one heme b, respectively. We used cryo-electron microscopy to obtain structural insights and combined these findings with biochemical studies and quantum mechanics/molecular mechanics (QM/MM) modelling to explore the limits of C-H bond activation by molybdenum-dependent hydroxylases. Taken together, the studies performed on both enzymes provide further insights into the mechanisms of water-dependent hydrocarbon activation in anoxic environments.
1Rabus, R. et al., J Mol Microbiol Biotechnol, 26, 5-28 (2016)
2Strijkstra, A. et al., AEM, 80, 7592–7603 (2014)
3Aeckersberg, F. et al., Microbiol, 156, 5-14 (1991)
4Callaghan A., Front Microbiol, 4, 1-9 (2013)