Switching an Artificial P450 Peroxygenase into Peroxidase via Mechanism-Guided Protein Engineering

Abstract

Exploring the catalytic promiscuity of enzymes is a longstanding challenge and a current topic of interest. Our group previously modified a cytochrome P450BM3 monooxygenase to perform peroxygenase activity with assistance from a rationally designed dual-functional small molecule (DFSM). However, the DFSM-facilitated P450-H2O2 system showed limited peroxidase activity. On the basis of a mechanistic analysis of the possible competitive oxidation pathways, the present work applies a protein engineering strategy of mutating redox-sensitive residues that enables the peroxygenase system to achieve efficient peroxidase activity. The engineered system exhibits efficient one-electron oxidation activity toward various substrates, including guaiacol, 2,6-dimethoxyphenol, o-phenylenediamine, and p-phenylenediamine. This system attains the best peroxidase activity of any P450 reported to date and rivals most natural peroxidases, suggesting significant potential for practical applications. This work provides insights and strategies relevant for expanding the catalytic promiscuity of P450s through combining the effects of protein engineering and exogenous molecules.

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

“…In more recent studies, the effect of DFSM ( i. e ., Im‐C6‐Phe) on P450BM3 enzyme activity and enantioselectivities to synthesize ( R )‐styrene oxide has been further optimized by combination with enzyme engineering [78] . With the DFSM strategy, different P450BM3 variants displayed high O ‐demethylation on aromatic ethers [79] and peroxidase activity on guaiacol, 2,6‐dimethoxyphenol, o ‐phenylenediamine, and p ‐phenylenediamine [80] . Based the crystal structure of the P450BM3‐F87A mutant in complex with Im‐C6‐Phe and NH 2 OH, multiple polar interactions and hydrophobic interactions were identified between the enzyme and the anchor group of DFSM (Figure 9B) [81] .…”

“…[78] With the DFSM strategy, different P450BM3 variants displayed high O-demethylation on aromatic ethers [79] and peroxidase activity on guaiacol, 2,6-dimethoxyphenol, o-phenylenediamine, and pphenylenediamine. [80] Based the crystal structure of the P450BM3-F87A mutant in complex with Im-C6-Phe and NH 2 OH, multiple polar interactions and hydrophobic interactions were identified between the enzyme and the anchor group of DFSM (Figure 9B). [81] The computational study demonstrated that the H 2 O 2 maintained a strong H-bonding network with a bridging water and the imidazolyl group of the DFSM, which speeds up the Cpd I formation via a favored heterolytic OÀ O cleavage pathway, thus to affect the overall kinetics.…”

A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

“…Interestingly, the oxidation of guaiacol, a classical substrate of peroxidases [141][142][143][144], catalyzed by the DFSM-facilitated P450BM3-H 2 O 2 system yielded demethylated catechol as a major product, suggesting it mainly functioned as a peroxygenase but not as a peroxidase [94]. After carefully analyzing the catalytic mechanism of the potential competitive oxidation pathways in the DFSM-facilitated P450BM3-H 2 O 2 system, Ma et al hypothesized that mutation of redox-sensitive residues may enable switching of peroxygenase activity to peroxidase activity [145]. Using a semi-rational design approach, similar to FRISM (focused rational iterative site-specific mutagenesis) named by Reetz and Wu [146,147], Ma et al identified mutations of three key redox-sensitive tyrosine residues that are located on the surface of P450.…”

“…This suggests that protein engineering of the DFSM-facilitated P450 peroxygenase system may also have its own unique advantages for controlling reaction selectivity in comparison with natural NADPH-dependent P450s. In addition, the high peroxidase activity of the DFSM-facilitated P450-system developed recently expanded the catalytic promiscuity of the system [145], whose further application in organic transformation is expected.…”

Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

“…By mimicking the molecular structure and catalytic mechanism of natural peroxygenase and peroxidase [ 32 , 33 ], we recently developed a unique P450–H 2 O 2 system by employing an exogenous fatty acyl amino acid equipped with a terminal imidazolyl (called a dual-functional small molecule, DFSM) to activate the peroxide-shunt pathway of P450BM3. The typical DFSM, such as N -(ω-imidazolyl)-hexanoyl-l-phenylalanine (Im-C6-Phe), was thought to bind with P450BM3 by the acyl amino acid, as the anchor group and its imidazolyl moiety serves as the acid–base catalyst to facilitate H 2 O 2 activation (shown as the red arrow pathway in Figure 1 A) [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ]. This system uses various P450BM3 variants to efficiently catalyze a variety of monooxygenation and oxidation reactions towards non-native substrates, e.g., styrene epoxidation, thioanisole sulfoxidation, hydroxylation of alkanes and aromatic compounds, demethylation of aromatic ethers, as well as one-electron oxidation of phenol and aromatic amines ( Figure 1 B).…”