Hydrocarbon biosynthesis by non-conventional P450 enzymes

Fatty acid peroxygenases have been underscored in hydrocarbon biosynthesis due to their capacity to perform C-C scission en route, producing olefins, a central building block for the production of sustainable plastics, polymers, and fuels. These biocatalysts possess non-canonical and complex mechanisms, which involve redox partners, co-factors, hydrogen abstraction, controlled electrons and protons delivery that culminate in bifurcated chemoselectivity into hydroxylation or decarboxylation. Herein, we revealed different structural complexes of iso-functionally clustered decarboxylases bound to either saturated or unsaturated substrates, along with structure-guided protein engineering, molecular dynamics simulations and unsupervised machine learning. This mechanism is underpinned in concerted molecular arrangements involving the distal binding pocket, known as hydrophobic cradle, and substrate to orient the Cβ atom towards the catalytic heme-iron. We also demonstrated that the lack of the aromatic residue from the Phe-His-Arg triad, positioned at the heme proximal site, preserved the chemoselectivity for alkenes, underlining a distinct standpoint regarding the structural determinants for β regiochemistry. Taken together, these findings untangle key molecular factors governing the tunable biocatalytic selectivity of P450 peroxygenases that are central for the sustainable production of olefins from oleic acid, the most abundant and relevant renewable fatty acids in nature.