Characterization of the Orphan Cytochrome P450 CYP107J1 From Bacillus subtilis Through Peroxygenase Activity Engineering.

Cytochrome P450 monooxygenases (P450s) not only play many physiological roles in oxidative metabolism but are also promising biocatalysts for the synthesis of organic molecules. Bacillus subtilis strain 168, which possesses eight P450 genes, has been extensively studied as a model bacterium, but the catalytic function of CYP107J1 remains to be fully elucidated. Here, we investigated the catalytic function of CYP107J1. Because the genes encoding the reductase components of CYP107J1 could not be identified from the genome sequence, putidaredoxin (Pdx) and its reductase (PdR) were first used to reconstitute the enzymatic activity of CYP107J1. The enzyme showed oxidation activity toward 4-alkylbenzoic acids with a carbon chain length of 3-8, although the activity was low. To enhance the activity of CYP107J1, over that observed when using heterologous redox partners, we next engineered CYP107J1 into a hydrogen peroxide-driven form (i.e., peroxygenase). Only two amino acid substitutions in the active site of CYP107J1 were required for a peroxygenase variant exhibiting 28 times higher catalytic activity toward 4-hexylbenzoic acid than the wild-type enzyme supported by Pdx and PdR. This highly active enzyme enabled detailed characterization of CYP107J1. Interestingly, the engineered enzyme not only oxidized 4-alkylbenzoic acids but also efficiently produced the valuable dye indigo by simply mixing the enzyme with indole and hydrogen peroxide. Enzymes of the CYP107J subfamily are widely distributed among bacteria of genus Bacillus. The findings from this study will facilitate further exploitation of the catalytic potential of CYP107J subfamily enzymes.