Semirationally Engineering an Efficient P450 Peroxygenase for Regio- and Enantioselective Hydroxylation of Steroids

ACS Catalysis, Journal Year: 2025, Volume and Issue: unknown, P. 2977 - 2986

Published: Feb. 5, 2025

Language: Английский

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Emerging Strategies for Modifying Cytochrome P450 Monooxygenases into Peroxizymes

Accounts of Chemical Research, Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 31, 2024

ConspectusCytochrome P450 monooxygenase is a versatile oxidizing enzyme with great potential in synthetic chemistry and biology. However, the dependence of its catalytic function on nicotinamide cofactor NAD(P)H redox partner proteins limits practical application vitro. An alternative to expensive cofactors low-cost H2O2, which can be used directly exploit P450s. peroxide shunt pathway generally inefficient at driving catalysis compared normal NAD(P)H-dependent activity. Over last few decades, scientific community has made continuous efforts use directed evolution or site-directed mutagenesis modify monooxygenases into their peroxizyme modes─peroxygenase peroxidase. Despite significant progress, obtaining efficient peroxizymes remains huge challenge. Here, we summarize our modulate activity applications challenging selective C–H oxidation, oxygenation, oxyfunctionalization over past seven years. We first developed dual-functional small molecule (DFSM) strategy for transforming P450BM3 peroxygenase. In this strategy, typical DFSM, such as N-(ω-imidazolyl)-hexanoyl-l-phenylalanine (Im-C6-Phe), binds protein an anchoring group one end plays general acid–base role activation H2O2 imidazolyl other end. Compared O–O homolysis mechanism absence addition DFSM efficiently enables heterolytic cleavage adduct Fe–O–OH, thus being favored formation active species compound I, been demonstrated by combining crystallographic theoretical calculations. Furthermore, engineering showed unique performance DFSM-facilitated peroxygenase highly difficult oxidation bonds. This was during chemoselective hydroxylation gaseous alkanes, regioselective O-demethylation aryl ethers, (R)-enantioselective epoxidation styrene, regio- enantiomerically diverse alkylbenzenes. Second, that could effectively switched peroxidase mode through mechanism-guided redox-sensitive residues. Utilizing enabled direct nitration unsaturated hydrocarbons including phenols, aromatic amines, styrene derivatives, not only P450-catalyzed phenols amines time but also example biological olefins. Finally, report tunnel enable several different time, providing approach accessing engineered peroxygenases. Account, highlight emerging strategies have producing biocatalysts. Although primarily applied date, both residue show extend These expanded scope catalysis. Additionally, they provide solution inert bonds chemistry.

Language: Английский

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Unraveling the Catalytic Mechanism of Taxadiene-5α-hydroxylase from Crystallography and Computational Analyses

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(6), P. 3912 - 3925

Published: Feb. 26, 2024

Paclitaxel is a famous chemotherapeutic agent, but its microbial production poses long-standing challenge due to poor product selectivity. Taxadiene-5α-hydroxylase (CYP725A4) plays crucial role in the biosynthesis of paclitaxel, catalyzing oxidation taxadiene and iso-taxadiene. This process yields several products, including byproducts 5(12)-oxa-3(11)-cyclotaxane (OCT) 5(11)-oxa-3(11)-cyclotaxane (iso-OCT), as well target compound taxadien-5α-ol (T5OH). Despite extensive studies, molecular mechanism CYP725A4-catalyzed transformations still elusive, which could impede our understanding further engineering paclitaxel biosynthetic pathway. In this study, crystal structure CYP725A4 complex with elucidated. Through comprehensive computational analyses, catalytic mechanisms natural are deciphered. Our calculations indicate that affords zwitterion intermediate, can undergo two competing transformation routes. One involves formation epoxide, undergoes water-mediated rearrangement form T5OH product. alternative pathway, protonation oxygen intermediate facilitates subsequent hydride transfer carbon–oxygen coupling, resulting side products OCT/iso-OCT. Contrary taxadiene, hydroxylation at C5 iso-taxadiene directly T5OH. These crystallographic studies analyses have yielded valuable insights into laid foundation for CYP725A4.

Language: Английский

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Natural alternative heme-environments allow efficient peroxygenase activity by cytochrome P450 monooxygenases

Catalysis Science & Technology, Journal Year: 2023, Volume and Issue: 13(21), P. 6264 - 6273

Published: Jan. 1, 2023

Mild oxyfunctionalization reactions driven by hydrogen peroxide using cytochrome P450 monooxygenases with alternative heme environments.

Language: Английский

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Computational Modeling of the Enzymatic Achmatowicz Rearrangement by Heme-Dependent Chloroperoxidase: Reaction Mechanism, Enantiopreference, Regioselectivity, and Substrate Specificity

Journal of Chemical Information and Modeling, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 31, 2025

The chloroperoxidase from Caldariomyces fumago (CfCPO) catalyzes the oxidative ring expansion of α-heterofunctionalized furans via Achmatowicz rearrangement, providing an elegant tool to convert furan rings into complex-prefunctionalized scaffolds. However, mechanism this transformation remains unclear. Herein, CfCPO-catalyzed reaction rac-1-(2-furyl)ethanol (1a) is studied by quantum chemical calculations and molecular dynamics simulations. reveal that conversion follows general reaction. Notably, binding 1a enzyme's active site influences Compound I (Cpd I) formation, (R)-1a enantiomer results in a lower barrier compared (S)-1a, explaining observed (R)-enantiopreference toward racemic substrate. Additionally, due weaker steric hindrance between porphyrin substrate, nucleophilic attack Cpd on core preferred at less-substituted C4=C5 bond, rationale for experimentally regioselectivity. Finally, bottleneck residues substrate delivery channel also surroundings are proposed be responsible specificity CfCPO. This study lays theoretical foundation rational design new CPOs catalyze rearrangement with broader spectrum or specific stereopreference.

Language: Английский

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Structure–Function Analysis of the Self-Sufficient CYP102 Family Provides New Insights into Their Biochemistry

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(5), P. 2161 - 2161

Published: Feb. 28, 2025

Cytochromes P450 are a superfamily of heme-containing monooxygenases involved in variety oxidative metabolic reactions, primarily catalyzing the insertion an oxygen atom into C-H bond. CYP102 represents first example bacterial that can be classified as type II (eukaryotic-like) and functions catalytically self-sufficient enzyme. These unique features have made attractive system for studying structure function. However, overall picture specific amino acid residues crucial to functioning effect mutations on catalysis is yet reported. Such approach will aid protein engineering approaches used improve this To address research knowledge gap, we investigated 105 crystal structures study. We demonstrate active site highly dynamic flexible. Amino play critical roles substrate binding, orientation, anchoring were identified. Mutational studies highlighted acids provided possible bioengineering improvement strategies CYP102. Decoy molecules promising agent deceiving permitting non-native substrates site. Ru(II)-diimine photosensitizers zinc/cobalt (III) sepulchrate (Co(III)Sep) could alternative electron sources. The present study serves reference understanding structure-functional analysis family members precisely enzymes general. Significantly, work contributes effort develop improved enzyme, thereby advancing field potentially leading new industrial applications.

Language: Английский

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In situ insertion of one or two types of hydroxy-rich noncanonical amino acids into one protein

Process Biochemistry, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

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Unravelling the C-C and C-N coupling mechanism for the CYP96T1-catalyzed biosynthesis of Amaryllidaceae alkaloids

Molecular Catalysis, Journal Year: 2023, Volume and Issue: 550, P. 113609 - 113609

Published: Oct. 7, 2023

Language: Английский

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Revisiting strategies and their combinatorial effect for introducing peroxygenase activity in CYP102A1 (P450BM3)

Molecular Catalysis, Journal Year: 2024, Volume and Issue: 557, P. 113953 - 113953

Published: Feb. 22, 2024

Different strategies have previously been reported to convert cytochrome P450 monooxygenases peroxygenases, allowing H2O2-driven oxyfunctionalization reactions. Comparison of the BM3 (CYP102A1) peroxygenase variant 21B3, obtained through enlargement active site by F87A mutation followed mutational stabilization towards H2O2, with BM3_T268E variant, an acid-base catalyst introduced, showed 21B3 be superior peroxygenase. A combination these two (21B3_T268E combinatorial mutant), however, resulted in reduced activity. The further introduction F(A)87V and A328F mutations (87–328 variants), improve regioselectivity on n-alkanes, a loss activity dodecanoic acid, substrate commonly used evaluate BM3. Although reduction was observed for styrene, mutants yielded higher enantioselectivities R-styrene oxide. α-olefins were, comparable between different variants BM3, but absolute selectivity epoxidation 21B3_87-328 21B3_T268E_87-382. Structural investigation architecture significant differences I-helix heme accessibility. According results, future directed evolution studies will require selective pressure not only also specific activated rather than surrogate substrates.

Language: Английский

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Tuning the peroxidase activity of artificial P450 peroxygenase by engineering redox-sensitive residues

Faraday Discussions, Journal Year: 2024, Volume and Issue: 252, P. 52 - 68

Published: Jan. 1, 2024

Engineering redox-sensitive residues dramatically increases the peroxidase activity of P450BM3 monooxygenase, providing new insights and a strategy for regulating P450s' promiscuous functionality.

Language: Английский

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