Directed Evolution of Protoglobin Optimizes the Enzyme Electric Field

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(24), P. 16670 - 16680

Published: June 7, 2024

To unravel why computational design fails in creating viable enzymes, while directed evolution (DE) succeeds, our research delves into the laboratory of protoglobin. DE has adapted this protein to efficiently catalyze carbene transfer reactions. We show that previously proposed enhanced substrate access and binding alone cannot account for increased yields during DE. The 3D electric field entire active site is tracked through dynamics, clustered using affinity propagation algorithm, subjected principal component analysis. This analysis reveals notable changes with DE, where distinct topologies influence transition state energetics mechanism. A chemically meaningful emerges takes lead facilitates crossing barrier transfer. Our findings underscore intrinsic dynamic's on enzyme function, ability switch mechanisms within same protein, crucial role design.

Language: Английский

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

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(10), P. 7893 - 7900

Published: May 6, 2024

Cytochrome P450 enzymes play a pivotal role in biosynthetic and metabolic transformations. Especially, cytochrome reductase (CPR) acts as the key electron donor for oxygen activation by monoxygenases, but transfer mechanism within CPR is largely elusive. Here, extensive molecular dynamics (MD) quantum mechanics/molecular mechanics (QM/MM) calculations were performed to elucidate CPR's mechanism. We found that from FADH– FMN occurs through proton-coupled (PCET) Glu142 transfers proton via two-water-molecule chain, concurrent with FMN. The subsequent ET FADH• FMNH• involves an Asp675-mediated PCET process, where Ser457-assisted Asp675 coupled FMNH•. Notably, local electric field doubly protonated His180 significantly enhances reactions both kinetically thermodynamically. This study highlights vital of facilitating biological enzymatic reactions.

Language: Английский

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Understanding the Scope of Cytochrome P450-Catalyzed Radical Dimerization of Diketopiperazines

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

Published: Jan. 2, 2025

DtpC was isolated from the ditryptophenaline biosynthetic pathway found in filamentous fungi as a cytochrome P450 (P450) that catalyzes dimerization of diketopiperazines. More recently, several similar P450s were discovered. While vast majority such generate asymmetric diketopiperazine dimers, and other fungal predominantly catalyze formation symmetric dimer products. Dimeric compounds can have interesting biological activities, mode substantially affect their bioactivities substantially. Here, we set out to examine mechanism scope catalyzed by using both chemically modified substrate molecules mutants selected screening randomly mutated recombinant variants. Use N1- N10-methylated substrates supports proposal initial radical occurs extraction N1 indole nitrogen for this dimerase. Further vitro studies revealed capable accepting range structurally variable substrates, including N-demethylated diketopiperazines, forming homo- heterodimeric Moreover, introduction single mutations identified through random at around substrate-binding pocket led conversion into catalyst generated dimers various The versatility serve good starting point directed evolution versatile catalysts generation not only diketopiperazines derived standard nonstandard amino acids but also possibly more divergent analogs

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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Mechanistic Insights into CYP199A4-Catalyzed α-Hydroxyketone Formation and Hydrogen Bond-Assisted C–C Bond Cleavage Catalyzed by the CYP199A4 F182L Mutant

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(4), P. 1526 - 1526

Published: Feb. 11, 2025

CYP199A4 is a cytochrome P450 and can catalyze the hydroxylation of 4-propionylbenzoic acid (4-pIBA) to generate α-hydroxyketone with high stereoselectivity. The F182L mutant (F182L-CYP199A4) has been shown support cleavage C-C bond between carbonyl hydroxyl groups α-hydroxyketone, whereas wild-type cannot. To uncover how Phe182 regulates substrate reactivity, we conducted classical molecular dynamics (MD) quantum mechanics/molecular mechanics (QM/MM) MD simulations on these systems. results predicted that formation preferentially led (S)-enantiomer. Moreover, findings revealed F182L-CYP199A4 facilitated hydrogen reactive peroxoanion (POA) species. This interaction stabilized near POA promoted subsequent cleavage. mechanism were elucidated by employing hybrid density functional theory (DFT). involved C-H 4-pIBA rate-limiting energy barrier 17.1 kcal/mol. catalyzed occurred via radical attack mechanism.

Language: Английский

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Mechanism of the Oxidative Ring-Closure Reaction during Gliotoxin Biosynthesis by Cytochrome P450 GliF

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(16), P. 8567 - 8567

Published: Aug. 6, 2024

During gliotoxin biosynthesis in fungi, the cytochrome P450 GliF enzyme catalyzes an unusual C-N ring-closure step while also aromatic ring is hydroxylated same reaction cycle, which may have relevance to drug synthesis reactions biotechnology. However, as details of mechanism are still controversial, no applications been developed yet. To resolve and gain insight into steps leading ring-closure, we ran a combination molecular dynamics density functional theory calculations on structure reactivity tested range possible mechanisms, pathways models. The show that, rather than hydrogen atom transfer from substrate Compound I, initial proton transition state followed by fast electron en route radical intermediate, hence non-synchronous abstraction takes place. intermediate then reacts OH rebound form biradical through between centers, gives products. Interestingly, energetics mechanisms appear little affected addition polar groups model predict that can be catalyzed other isozymes bind substrate. Alternative pathways, such pathway starting with electrophilic attack arene epoxide, high energy ruled out.

Language: Английский

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Molecular Basis for the P450-Catalyzed sp³ C–N Glycosidic Bond Formation in Staurosporine Biosynthesis

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(19), P. 14274 - 14284

Published: Sept. 12, 2024

Language: Английский

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Machine-Learning Prediction of Protein Function from the Portrait of Its Intramolecular Electric Field

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 7, 2024

We introduce a machine learning framework designed to predict enzyme functionality directly from the heterogeneous electric fields inherent protein active sites. apply this method curated data set of heme-iron oxidoreductases, spanning three classes: monooxygenases, peroxidases, and catalases. Conventional analysis, focused on simplistic, point along Fe–O bond, is shown be inadequate for accurate activity prediction. Our model demonstrates that enzyme's 3-D field, alone, can accurately its function, without relying additional protein-specific information. Through feature selection, we uncover key field components not only validate previous studies but also underscore crucial role multiple beyond traditionally emphasized bond in heme enzymes. Furthermore, by integrating dynamics, principal component clustering, QM/MM calculations, reveal while dynamic complexities structures obscure predictions, still retains accuracy. This research significantly advances our understanding how scaffolds possess signature tailored their functions at site. Moreover, it presents novel electrostatics-based tool harness these predicting function.

Language: Английский

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Bacterial acyl homoserine lactones triggered non‐native substrate hydroxylation catalysed by directed‐evolution‐derived cytochrome P450BM3 mutants

ChemCatChem, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 14, 2024

Abstract We report the directed evolution of cytochrome P450BM3 to efficiently utilize bacterial quorum sensing signalling molecule N ‐decanoyl homoserine lactone (C10‐HSL) as an effective decoy molecule. This represents first important step in our endeavor develop a self‐sufficient decoy‐molecule system whole‐cells that only necessitates addition culture medium and substrate realize hydroxylation non‐native substrates. Following five rounds evolution, mutant P450BM3, presence C10‐HSL, catalyzed benzene at rate 475 min −1 , highest turnover recorded for any P450 enzyme, achieving 46% yield whole‐cell reaction system. High‐resolution X‐ray crystal structure analysis series mutants narrates process, revealing how C10‐HSL is fixed binding pocket permit Finally, introduction synthase gene ExpI into Escherichia coli enabled situ production realizing, time, substrates without need laborious synthesis molecules.

Language: Английский

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A nucleobase-driven P450 peroxidase system enables regio- and stereo-specific formation of C─C and C─N bonds

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(46)

Published: Nov. 7, 2024

P450 peroxidase activities are valued for their ability to catalyze complex chemical transformations using economical H 2 O ; however, they have been largely underexplored compared monooxygenase and peroxygenase activities. In this study, we identified an unconventional enzyme, PtmB, which catalyzes the dimerization of purine nucleobases tryptophan-containing diketopiperazines (TDKPs), yielding C3-nucleobase pyrroloindolines nucleobase-TDKP dimers. Unlike typical TDKP enzymes reliant on NAD(P)H cofactors electron transfer systems, its analogs exhibit remarkable activity in synthesizing adenine other modified 6-aminopurine Structural analysis PtmB–substrate complex, mutation assays, computational investigations reveal adenine’s dual role as both substrate acid–base catalyst activating generate Compound I (Cpd I). This initiates a specific radical cascade reaction, facilitating formation precise C─C C─N bonds. Biochemical assays molecular dynamics simulations demonstrate that 6-NH hydrogen-bonding networks induce necessary conformational changes activation, thereby driving activity. study unveils unusual catalytic mechanism system underscores pivotal enzyme-mediated reactions, offers different prospects developing peroxidases nucleobase-based biocatalysts.

Language: Английский

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