Hijacking Chemical Reactions of P450 Enzymes for Altered Chemical Reactions and Asymmetric Synthesis

Abstract

Cytochrome P450s are heme-containing enzymes capable of the oxidative transformation of a wide range of organic substrates. A protein scaffold that coordinates the heme iron, and the catalytic pocket residues, together, determine the reaction selectivity and regio- and stereo-selectivity of the P450 enzymes. Different substrates also affect the properties of P450s by binding to its catalytic pocket. Modulating the redox potential of the heme by substituting iron-coordinating residues changes the chemical reaction, the type of cofactor requirement, and the stereoselectivity of P450s. Around thousands of P450s are experimentally characterized, therefore, a mechanistic understanding of the factors affecting their catalysis is increasingly vital in the age of synthetic biology and biotechnology. Engineering P450s can enable them to catalyze a variety of chemical reactions viz. oxygenation, peroxygenation, cyclopropanation, epoxidation, nitration, etc., to synthesize high-value chiral organic molecules with exceptionally high stereo- and regioselectivity and catalytic efficiency. This review will focus on recent studies of the mechanistic understandings of the modulation of heme redox potential in the engineered P450 variants, and the effect of small decoy molecules, dual function small molecules, and substrate mimetics on the type of chemical reaction and the catalytic cycle of the P450 enzymes.

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

“…Given the attractive features of P450 BM3, the enzyme has garnered significant attention and has been the subject of numerous reviews highlighting its exceptional properties and characteristics. Reviews have been conducted on various aspects of P450 BM3, including structural and mechanistic details, − hydroxylation, ,,− pharmaceutical compound hydroxylation, ,− epoxidation, ,,, and carbene/nitrene transfer. ,,,,,,− Additionally, studies have explored the use of decoy molecules to improve enzymatic activity, ,,,,,,− as well as P450 BM3-inorganic complexes. − Reviews of P450 BM3 frequently place it within a broader context of other P450 enzymes, resulting in a lack of focus. Though some reviews do describe reactions catalyzed by P450 BM3 variants, they are typically limited to discussion of the “best” variants for a given reaction and do not offer a comprehensive review of reported variants.…”

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

“…CYPs act as monooxygenases and catalyze more than 40 different chemically challenging reactions including hydroxylation of inactive CH bond, epoxidation, C−C bond cleavage, dehalogenation and so on [3,4,9–13] . In addition to the broad range reaction spectrum, P450 monooxygenases accept a variety of substrates from different chemical groups such as fatty acids, terpenes, steroids, aromatic compounds, vitamins, antibiotics, pharmaceuticals, and pesticides [12,14] .…”

“…Although P450s are powerful tools for industry, owing to accepting various substrates and catalyzing a diverse range of reactions, there are several drawbacks to their large‐scale industrial applications. These limitations are low catalytic activity towards non‐natural substrates, low stability in organic solvents, requirement of expensive nicotinamide cofactor and its efficient regeneration, low substrate solubility in aqueous media, and low coupling efficiency [7,12,13,19,20] . In addition, efficient heterologous expression of P450s as active protein and complicated protein purification process are other limitations that hinder widespread industrial application of P450s [21]…”

“…The cytochrome P450, heme-thiolate containing superfamily (P450s or CYPs; EC 1.14.13.x, EC 1.14.14.x, EC 1.14.15.x) is one of the oldest and largest gene families.. [1][2][3] CYPs are present in almost all living organisms and play important roles in primary and secondary metabolism such as natural product biosynthesis, drug metabolism, and xenobiotic/pollutant degradation.. [1,[4][5][6][7][8] CYPs act as monooxygenases and catalyze more than 40 different chemically challenging reactions including hydroxylation of inactive CH bond, epoxidation, CÀ C bond cleavage, dehalogenation and so on. [3,4,[9][10][11][12][13] In addition to the broad range reaction spectrum, P450 monooxygenases accept a variety of substrates from different chemical groups such as fatty acids, terpenes, steroids, aromatic compounds, vitamins, antibiotics, pharmaceuticals, and pesticides. [12,14] Furthermore, biotransformation products of these compounds are further used for the production of various products in pharmaceutical, cosmetic, polymer, and adhesive industries.…”

“…These limitations are low catalytic activity towards non-natural substrates, low stability in organic solvents, requirement of expensive nicotinamide cofactor and its efficient regeneration, low substrate solubility in aqueous media, and low coupling efficiency. [7,12,13,19,20] In addition, efficient heterologous expression of P450s as active protein and complicated protein purification process are other limitations that hinder widespread industrial application of P450s. [21] Most of the P450 monooxygenases require an external electron transfer protein for catalytic activity and use NAD(P)H as an electron donor.…”