Phenoxazinone Synthase-Like Activity of Rationally Designed Heme Enzymes Based on Cytochrome P450bm3

Abstract

The development of functional metalloenzymes holds significant promise for the biosynthesis of valuable chemicals, including phenoxazinones, which are catalyzed by native phenoxazinone synthase (PHS). In this study, we harnessed a H2O2-dependent catalytic system to convert aromatic amine substrates to phenazines and phenoxazinones using P450BM3 in conjunction with dual-functional small molecules (DFSMs). The modification of amino acids at 78, 87, and 264 positions within the heme active site notably influenced the catalytic activity of the system. Specifically, the F87G/V78A mutant exhibited a remarkable kcat/Km of 175.69 mM-1s-1 for the oxidation of 2, 5-diaminobenzenesulfonic acid (2,5-DABSA), while the F87G mutant displayed superior catalytic efficiencies for o-aminophenol (OAP) and o-phenylenediamine (OPD) oxidation, with kcat/Km values of 276.92 mM-1s-1 and 79.13 mM-1s-1, respectively. These values surpass those of widely used enzymes such as laccase, dye-decolorizing peroxidase, o-aminophenol oxidase and myoglobin. Overall, this study demonstrates that this enzymatic catalytic system offers an environmentally friendly alternative to traditional chemical method for aromatic amines oxidation, with broad potential applications in the synthesis of heterocyclic compounds with broad applications in pharmaceuticals, materials science, and green chemistry.

Keywords: Cytochrome P450BM3BiocatalysisphenazinePhenoxazinone