Evolving a P450BM3 Peroxygenase for the Production of Indigoid Dyes from Indoles

Abstract

Herein, we describe an environmentally benign enzymatic approach for the preparation of indigoid from indole derivatives. A series of beneficial P450BM3 mutants were obtained using a stepwise approach involving site‐directed, random, and combinatory hot‐site mutations. Using H2O2 as the terminal oxidant and N‐(ω‐imidazolyl)‐hexanoyl‐L‐phenylalanine as a co‐catalyst, the quadruple‐mutant F87G/T268V/F77I/E140D efficiently catalyzed the 3‐hydroxylation of indole and subsequent autooxidation to indigo with higher efficiency than the flavin‐containing monooxygenase, PTDH‐mFMO, which was the best oxidizing enzyme for indigo synthesis reported to date. Following this procedure, 12 indigoid compounds were prepared using indole derivatives with various substituents as starting materials with moderate to high isolated yields (31.3–79.5 %). The catalytic efficiencies (kcat/Km) of the beneficial mutants for typical indole substrates ranged from 182–2849 mM−1 ⋅ min−1, almost 2‐ to 712‐fold higher than those of the reported P450 enzymes. This study provides a new enzymatic approach for the biosynthesis of indigoid dyes from indole derivatives.

Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

“…Interestingly, certain mutation sites in P450 peroxygenases obtained through directed evolution were correlated with the corresponding water tunnels predicted by CAVER. For example, the peroxygenase-improved mutants GV/G315C, GV/L277M, GV/R190Q, and GV/F77I derived from the GV mutant of P450BM3 via directed evolution, included four mutation sites (G315, L277, R190, and F77, respectively), all of which were involved in the water tunnels predicted by CAVER (Figure S11, Table S14). Moreover, when we used CAVER to analyze the water tunnels of P450BM3_F87A, i.e., the parent enzyme of 21B3, which is a well-known P450 peroxygenase prepared by Arnold and co-workers via directed evolution, , we discovered that four of the nine key mutation sites in 21B3 were located at the bottleneck of water tunnels (Figure B).…”

Emerging Strategies for Modifying Cytochrome P450 Monooxygenases into Peroxizymes

“…In addition, the evolved P450BM3 peroxygenase variants selectively catalyzed 3-hydroxylation of indole to produce indigo following spontaneous oxidative condensation in the presence of Im-C6-Phe. 52 The most effective mutant (F87G/T268 V/F77I/E140D) yielded a catalytic TON of 6588. This was higher than the best reported oxidase for indigo synthesis, the flavin monooxygenase PTDH-mFMO.…”

“…DFSM-facilitated P450 peroxygenase can also catalyze the aromatic hydroxylation of benzene and naphthalene to produce phenol and 1-naphthol with moderate TONs, , respectively. In addition, the evolved P450BM3 peroxygenase variants selectively catalyzed 3-hydroxylation of indole to produce indigo following spontaneous oxidative condensation in the presence of Im-C6-Phe . The most effective mutant (F87G/T268 V/F77I/E140D) yielded a catalytic TON of 6588.…”

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

“…It has been the topic of extensive research over the past three decades and numerous laboratories have worked on evolving this enzyme to generate novel enzymatic properties and features. As a result, P450 BM3 and its variants have been shown to catalyze a wide variety of reactions, including hydroxylation, − epoxidation, − carbene transfer, − and nitrene transfer, − with a diverse range of substrates. The advances reported by the group of Frances Arnold on the evolution of P450 BM3 toward novel biocatalytic reactions, which were prominently featured in her 2018 Nobel-winning research, are among the most transformative.…”