The heme enzymes of the cytochrome P450 superfamily (CYPs) catalyse the selective hydroxylation of unactivated C-H bonds in organic molecules. There is great interest in applying these enzymes as biocatalysts with a focus on self-sufficient CYP 'fusion' enzymes, comprising a single polypeptide chain with the electron transfer components joined to the heme domain. Here we elucidate the function of the self-sufficient CYP116B46 fusion enzyme, from the thermophilic bacterium Tepidiphilus thermophilus. We demonstrate that it efficiently hydroxylates aromatic organic acids, exemplified by oxidation of 2-hydroxyphenylacetic acid to homogentisic acid (2,5-dihydroxyphenylacetic acid), an important metabolite in bacterial catabolism. In line with the thermophilic nature of the source bacterium, activity increased at higher temperatures, (50 °C), with a catalytic preference for NADPH over NADH. While self-sufficient fusion enzymes simplify biocatalysis
engineered peroxygenase activity is also a key advance in the application of these enzymes as biocatalysts as it eliminates the need for electron transfer partner proteins and nicotinamide cofactors. We demonstrate that a T278E mutation in the heme domain of CYP116B46, confers peroxygenase activity. This engineered peroxygenase enzyme is stable to elevated temperatures and catalytic concentrations of hydrogen peroxide, with an observed optimal activity resulting in a total turnover number of ~650.