CYP153A71 from Alcanivorax dieselolei: Oxidation beyond Monoterminal Hydroxylation of n-Alkanes

Abstract

Selective oxyfunctionalization of non-activated C–H bonds remains a major challenge in synthetic chemistry. The biocatalytic hydroxylation of non-activated C–H bonds by cytochrome P450 monooxygenases (CYPs), however, offers catalysis with high regio- and stereoselectivity using molecular oxygen. CYP153s are a class of CYPs known for their selective terminal hydroxylation of n-alkanes and microorganisms, such as the bacterium Alcanivorax dieselolei, have evolved extensive enzymatic pathways for the oxyfunctionalization of various lengths of n-alkanes, including a CYP153 to yield medium-chain 1-alkanols. In this study, we report the characterization of the terminal alkane hydroxylase from A. dieselolei (CYP153A71) for the oxyfunctionalization of medium-chain n-alkanes in comparison to the well-known CYP153A6 and CYP153A13. Although the expected 1-alkanols are produced, CYP153A71 readily converts the 1-alkanols to the corresponding aldehydes, fatty acids, as well as α,ω-diols. CYP153A71 is also shown to readily hydroxylate medium-chain fatty acids. The X-ray crystal structure of CYP153A71 bound to octanoic acid is solved, yielding an insight into not only the regioselectivity, but also the binding orientation of the substrate, which can be used in future studies to evolve CYP153A71 for improved oxidations beyond terminal n-alkane hydroxylation.