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 hundreds 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.

Keywords: asymmetric synthesis; biocatalysis; cytochrome P450; enzyme engineering; prosthetic group; regioselectivity; stereoselectivity.

Figure 1

The catalytic pocket of the representative P450 (P450_BM3) enzyme and the schematic representation of heme prosthetic groups present in the P450 and non-enzyme proteins. (A) Protoporphyrin IX, the prosthetic group of Cytochrome P450 (purple sticks), interacts with Trp96, Arg398, and Lys69 (Cyan sticks). Cys400 (yellow sticks) is a highly conserved residue that forms the heme–thiolate bond. Phe393 is present within the heme-binding motif of P450_BM3, and positioned close to the Cys400, and is represented by cyan sticks. (B,C) Comparison of different heme groups. Heme b is the prosthetic group in cytochrome P450 and myoglobin, whereas heme c is the prosthetic group in Cytochrome C.

Figure 2

The schematic representation of the catalytic cycle of cytochrome P450. (A) The species (Compound 0 and Compound 1) are indicated. The species’ redox potential (mV) is given in the bracket. The axial heme ligand (cysteine thiolate, indicated as an S-atom linked to iron) and distal ligand (a water molecule that changes as the cycle progresses) are also indicated. (B) Uncoupling reactions with different pathways leading to the collapse of the oxy-intermediate are indicated. The oxy-ferrous form can reform the ferric state by superoxide formation via autoxidation shunt pathway. ‘Compound 0’ disintegrates with the production of peroxide and forms the ferric state (Substrate Bound) through peroxide shunt pathway. Compound 1 collapses by double reduction and diprotonation leading to the production of water via oxidase shunt pathway. These collapses can occur if there is no timely delivery of electron/proton or inappropriate positioning of the substrate.

Figure 3

Comparison of heme redox potentials of wildtype and variants of P450_BM3. Redox potentials (mentioned in brackets) of NADPH, substrate-free and bound heme groups, and the oxy-ferrous states of (A) wild-type and (B) the variants F393A and F393H of P450_BM3. Redox potentials in mV (mentioned in brackets) of NADPH, dithionite, and substrate-free heme groups of (C) wild-type and (D) the variant C400S of P450_BM3. SHE: Standard Hydrogen Electrode.

Figure 4

Engineering of P450s for C=C and C-H bond functionalization through abiotic transfer reactions. (A) The natural reaction of P450 by oxene transfer. (B) Carbene and (C) Nitrene transfer. (D) Aziridination with azide nitrene sources catalyzed by the variant of P450.

Figure 5

Cyclopropanation stereoselectivity of variants of P450_BM3. The hemin and the variant T268A exhibited trans selectivity with a cis:trans ratio of 6:94 and 1:99, respectively. The C400S variant displays a strong preference for cis product formation.

Figure 6

Role of the substrate and the acid-base pair in H₂O₂-dependent peroxidation by P450 enzymes. (A) Salt-bridge formation between Arg242 (cyan sticks) and Fatty Acid (magenta sticks) in P450BSβ (PDB ID: 1IZO). (B) Rationally engineered acid-base pair in P450SPα. The variant was engineered by A245E substitution. Interactions of E245 (orange sticks) with Arg241 (cyan sticks) are represented in broken green lines (PDB ID: 3VOO). The heme prosthetic group is shown with yellow stick representation.

Figure 7

Schematics of different strategies used to tune the P450 enzymes to catalyze the H₂O₂-dependent oxidation reactions. (A) Strategy for oxidation of nonnatural substrates in the presence of decoy molecule by Cytochrome P450. This involves the addition of a dummy substrate to accommodate non-natural substrate by remolding the active site. (B) DFSM co-catalysis strategy that involves the addition of a co-catalyst to aid the NADPH-dependent Cytochrome P450 for activation of the peroxide pathway for the catalysis of reactions of non-native substrates.

Figure 8

Summary of the review. Strategies for hijacking P450s for non-natural properties to alter the chemical reactions and for the asymmetric synthesis. Altering hem redox potential for non-native reactions and to switch the stereoselectivity, and employing decoy molecules and DFSM catalysts for non-native reactions and/ or for accepting non-native substrates.