Local Electric Fields Drives the Proton-Coupled Electron Transfer within Cytochrome P450 Reductase

Li, Ningning; Yan, Shengheng; Wu, Peng; Li, Junfeng; Wang, Binju

doi:10.1021/acscatal.4c02215

Cited by 4 publications

(1 citation statement)

References 115 publications

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“…So far, many strategies have been developed to optimize the electron transfer process in P450s catalytic systems to improve the catalytic efficiency of P450s, and calculations such as molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) are used to elucidate the electron transfer mechanism of P450s [ 150 ]. These include an adequate supply of electrons for the catalytic system, the selection and modification of P450s and CRP through rational/semi-rational design and directed evolution, the construction of photocatalysis and electrocatalysis systems, etc.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Engineering Electron Transfer Pathway of Cytochrome P450s

He,

Liu,

2024

Molecules

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Cytochrome P450s (P450s), a superfamily of heme-containing enzymes, existed in animals, plants, and microorganisms. P450s can catalyze various regional and stereoselective oxidation reactions, which are widely used in natural product biosynthesis, drug metabolism, and biotechnology. In a typical catalytic cycle, P450s use redox proteins or domains to mediate electron transfer from NAD(P)H to heme iron. Therefore, the main factors determining the catalytic efficiency of P450s include not only the P450s themselves but also their redox-partners and electron transfer pathways. In this review, the electron transfer pathway engineering strategies of the P450s catalytic system are reviewed from four aspects: cofactor regeneration, selection of redox-partners, P450s and redox-partner engineering, and electrochemically or photochemically driven electron transfer.

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Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Engineering Electron Transfer Pathway of Cytochrome P450s

He,

Liu,

2024

Molecules

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Enhancing the specific activity of 3α-hydroxysteroid dehydrogenase through cross-regional combinatorial mutagenesis

Ma,

Li,

Yan

et al. 2024

International Journal of Biological Macromolecules

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Understanding the Directed Evolution of a Natural-like Efficient Artificial Metalloenzyme

Mukherjee,

Roy

2024

J. Phys. Chem. B

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The artificial metalloenzyme containing iridium in place of iron along with four directed evolution mutations C317G, T213G, L69V, and V254L in a natural cytochrome P450 presents an important milestone in merging the extraordinary efficiency of biocatalysts with the versatility of small molecule chemical catalysts in catalyzing a new-to-nature carbene insertion reaction. This is a show-stopper enzyme, as it exhibits a catalytic efficiency similar to that of natural enzymes. Despite this remarkable discovery, there is no mechanistic and structural understanding as to why it displays extraordinary efficiency after the incorporation of the four active site mutations by directed evolution methods, which so far has been intractable to any experimental methods. In this study, we have deciphered how directed evolution mutations gradually alter the protein conformational ensemble to populate a catalytically active conformation to boost a multistep catalysis in a natural-like artificial metalloenzyme using large-scale molecular dynamics simulations, rigorous quantum chemical (QM), and multiscale quantum chemical/molecular mechanics (QM/MM) calculations. It reveals how evolution precisely positions the cofactor−substrate in an unusual but effective orientation within a reshaped active site in the catalytically active conformation stabilized by C−H•••π interactions from more ordered mutated L69V and V254L residues to achieve preferential transition state stabilization compared to the ground state. This work essentially tracks down in atomistic detail the shift in the conformational ensemble of the highly active conformation from the less efficient single mutant to the most efficient quadruple mutant and offers valuable insights for designing better enzymes. The active conformation correctly reproduces the experimental barrier height and also accounts for the catalytic effect, which is in good agreement with experimental observations. Moreover, this conformation features an unusual bonding interaction in a metal-carbene species that preferentially stabilizes the rate-determining formation of an iridium porphyrin carbene intermediate to render the observed high catalytic rate acceleration. Our study provides crucial insights into the underlying rationale for directed evolution, reports the major catalytic role of nonelectrostatic interactions in enzyme catalysis different from the electrostatic model, and suggests a crucial principle toward designing enzymes with natural efficiency.

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Local Electric Fields Drives the Proton-Coupled Electron Transfer within Cytochrome P450 Reductase

Cited by 4 publications

References 115 publications

Engineering Electron Transfer Pathway of Cytochrome P450s

Engineering Electron Transfer Pathway of Cytochrome P450s

Enhancing the specific activity of 3α-hydroxysteroid dehydrogenase through cross-regional combinatorial mutagenesis

Understanding the Directed Evolution of a Natural-like Efficient Artificial Metalloenzyme

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