The substrate specificity in the <i>O</i>-demethylation of 4-alkylguaiacols by cytochrome P450 AgcA<sub>P450</sub>

Santos, Sonia; Bommareddy, Rajesh Reddy; Black, Gary W.; Singh, Warispreet; Huang, Meilan

doi:10.1039/d3cy00123g

Cited by 4 publications

(2 citation statements)

References 52 publications

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“…The results described here support molecular dynamics (MD) simulations that show closing of the active site of GcoA in response to substrate binding (2). Contrary to these observations, MD simulations predict AgcA (CYP255A1) prefers a closed conformation with smaller substrates but adopts an open conformation with larger substrates (39).…”

Section: Discussionsupporting

confidence: 72%

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

Harlington,

Das,

Shearwin

et al. 2024

Preprint

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TheO-demethylation of lignin aromatics is a rate-limiting step in their bioconversion to high-value compounds. A recently discovered cytochrome P450 enzyme SyoA was found to demethylate the sinapyl alcohol-derived (S-lignin) aromatic syringol. In this work, we solved high-resolution X-ray crystal structures of SyoA in the substrate-free and substrate-bound states and evaluate the demethylation ofpara-substituted S-lignin aromatics via the monooxygenase pathway and peroxide shunt pathway. We found that SyoA demethylates S-lignin aromatics with the following activity: 4-methylsyringol > syringaldehyde > syringol exclusively using the peroxide shunt pathway. The atomic-resolution structure of SyoA reveals the position of the non-canonical residues in the I-helix (Gln252 and Glu253). Site-directed mutagenesis of this amide-acid pair of a homologous CYP255 enzyme GcoA, which can catalyze the O-demethylation of guaiacol using both monooxygenase and peroxygenase activity, showed the amide-acid pair is critical for both pathways. This work expands the enzymatic toolkit for improving the capacity to funnel lignin towards high-value compounds, and defines the new chemistry within the active site of the enzyme that enables efficient peroxygenase activity. These insights provide a framework for engineered peroxygenase activity in other cytochrome P450 enzymes, with the potential for more facile catalysis, relative to traditional P450 monooxygenases which require difficult to handle redox partners and expensive nicotinamide cofactors.

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Section: Discussionsupporting

confidence: 72%

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

Harlington,

Das,

Shearwin

et al. 2024

Preprint

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“…The natural bond orbital (NBO) spin population was obtained at the UB3LYP/B2 level. The Grimme DFT-D3BJ corrections were added to all QM/MM calculations. − As both our calculations (refer to Tables S2 and S3) and previous studies show that S = 1/2 (doublet) and S = 3/2 (quartet) states exhibit generally similar reactivities for the Cpd I-mediated reactions, ,− our discussions are limited to the doublet state in the main text. The calculated spin populations of key atoms of the involved species are shown in Table S1.…”

Section: Methodsmentioning

confidence: 99%

Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

Zhou,

Feng,

Wang

et al. 2024

JACS Au

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P450 NascB catalyzes the coupling of cyclo-(l-tryptophan-l-proline) (1) to generate (−)-naseseazine C (2) through intramolecular C–N bond formation and intermolecular C–C coupling. A thorough understanding of its catalytic mechanism is crucial for the engineering or design of P450-catalyzed C–N dimerization reactions. By employing MD simulations, QM/MM calculations, and enhanced sampling, we assessed various mechanisms from recent works. Our study demonstrates that the most favorable pathway entails the transfer of a hydrogen atom from N7–H to Cpd I. Subsequently, there is a conformational change in the substrate radical, shifting it from the Re-face to the Si-face of N7 in Substrate 1. The Si-face conformation of Substrate 1 is stabilized by the protein environment and the π–π stacking interaction between the indole ring and heme porphyrin. The subsequent intermolecular C3–C6′ bond formation between Substrate 1 radical and Substrate 2 occurs via a radical attack mechanism. The conformational switch of the Substrate 1 radical not only lowers the barrier of the intermolecular C3–C6′ bond formation but also yields the correct stereoselectivity observed in experiments. In addition, we evaluated the reactivity of the ferric-superoxide species, showing it is not reactive enough to initiate the hydrogen atom abstraction from the indole NH group of the substrate. Our simulation provides a comprehensive mechanistic insight into how the P450 enzyme precisely controls both the intramolecular C–N cyclization and intermolecular C–C coupling. The current findings align with the available experimental data, emphasizing the pivotal role of substrate dynamics in governing P450 catalysis.

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Mechanistic Perspective on C–N and C–S Bond Construction Catalyzed by Cytochrome P450 Enzymes

Zhou,

Fan,

Zhang

et al. 2024

ACS Bio Med Chem Au

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The substrate specificity in the O-demethylation of 4-alkylguaiacols by cytochrome P450 AgcA_P450

Abstract: Alkylguaiacols are lignin-derived products obtained by reductive catalytic fractionation (RCF) of lignocellulosic biomass. Recently discovered AgcAP450 enzyme from CYP255A1 family oxidizes a range of 4-alkylguaiacols, having a preference for bulkier...

Cited by 4 publications

References 52 publications

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

Mechanistic Perspective on C–N and C–S Bond Construction Catalyzed by Cytochrome P450 Enzymes

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The substrate specificity in the O-demethylation of 4-alkylguaiacols by cytochrome P450 AgcAP450

Abstract: Alkylguaiacols are lignin-derived products obtained by reductive catalytic fractionation (RCF) of lignocellulosic biomass. Recently discovered AgcAP450 enzyme from CYP255A1 family oxidizes a range of 4-alkylguaiacols, having a preference for bulkier...

Cited by 4 publications

References 52 publications

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

Mechanistic Perspective on C–N and C–S Bond Construction Catalyzed by Cytochrome P450 Enzymes

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The substrate specificity in the O-demethylation of 4-alkylguaiacols by cytochrome P450 AgcA_P450