Mechanistic Insights into the Selective C–S Bond Formation by P450 TleB

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(14), P. 10658 - 10669

Published: July 1, 2024

The P450 monooxygenase TleB (CYP107E48) catalyzes intramolecular C–S bond formation in a thiol-containing substrate, yielding two sulfur-containing indolactam derivatives (P1 and P2). However, the key sites influencing TleB's product selectivity molecular mechanisms underlying selective are not fully understood. To address this, we created an artificial self-sufficient P450, TleB-CYP116B46, by fusing with reductase domain of CYP116B46. Structure-guided engineering TleB-CYP116B46 generates variant L85G 99% for P1 I282L/Q387L/I234F 95% P2. Exploring homologues generating corresponding mutants elucidate identified sites' crucial role selectivity. Computational studies suggest diradical mechanism both P2 products. Intriguingly, found that substrate radical could undergo conformational changes S–H indole groups. facilitates switch group, thereby leading to product. By contrast, barricades affording Our simulations highlight protein environment can dictate dynamics positioning radical, P450s.

Language: Английский

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Anchoring a Structurally Editable Proximal Cofactor‐like Module to Construct an Artificial Dual‐center Peroxygenase

Angewandte Chemie International Edition, Journal Year: 2023, Volume and Issue: 62(51)

Published: Sept. 15, 2023

Abstract A recent novel strategy for constructing artificial metalloenzymes (ArMs) that target new‐to‐nature functions uses dual‐functional small molecules (DFSMs) with catalytic and anchoring groups converting P450BM3 monooxygenase into a peroxygenase. However, this process requires excess DFSMs (1000 equivalent of P450) owing to their low binding affinity P450, thus severely limiting its practical application. Herein, structural optimization the DFSM‐anchoring group considerably enhanced by three orders magnitude ( K d ≈10 −8 M), approximating native cofactors, such as FMN or FAD in flavoenzymes. An cofactor‐driven peroxygenase was constructed. The co‐crystal structure bound DFSM clearly revealed precatalytic state which participates H 2 O activation, facilitating activity. Moreover, increased substantially decreases load equivalents while maintaining Furthermore, replacement showed disparate selectivity activity various substrates. This study provides an unprecedented approach assembling ArMs editable organic cofactors co‐catalytic center, thereby increasing promiscuity P450 enzymes.

Language: Английский

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A Simple Access to γ‐ and ε‐Keto Arenes via Enzymatic Divergent C─H Bond Oxyfunctionalization

Advanced Science, Journal Year: 2023, Volume and Issue: 10(34)

Published: Oct. 23, 2023

Abstract Performing divergent C─H bond functionalization on molecules with multiple reaction sites is a significant challenge in organic chemistry. Biocatalytic oxyfunctionalization reactions of these compounds to the corresponding ketones/aldehydes are typically hindered by selectivity issues. To address challenges, catalytic performance oxidoreductases explored. The results show that combining peroxygenase‐catalyzed propargylic oxidation Old Yellow Enzyme‐catalyzed reduction conjugated C─C triple bonds one‐pot enables regio‐ and chemoselective sp 3 distant from benzylic sites. This enzymatic approach yielded variety γ‐keto arenes diverse structural electronic properties yields up 99% regioselectivity 100%, which difficult achieve using other chemocatalysis enzymes. By adjusting bond, carbonyl group's position can be further tuned yield ε‐keto arenes. combined biocatalysts establish new synthetic pathways for accessing various challenging reactions.

Language: Английский

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Enantioselective High‐Throughput Assay Showcased for the Identification of (R)‐ as well as (S)‐Selective Unspecific Peroxygenases for C−H Oxidation

Angewandte Chemie International Edition, Journal Year: 2023, Volume and Issue: 62(46)

Published: Sept. 25, 2023

Identifying (bio)catalysts displaying high enantio-/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, high-throughput, stereoselective screening assay reported that gives information on enantioselectivity, stereopreference and activity as showcased peroxygenase-catalyzed hydroxylation. The based spectrophotometric analysis simultaneous formation NAD(P)H from alcohol dehydrogenase catalyzed enantioselective oxidation sec-alcohol product formed in peroxygenase reaction. was applied to investigate library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not yet. Thereby, previously non-described wild-type (S)- well (R)-stereoselectivity hydroxylation representative model substrates were identified, reaching up 98 % ee (R)- 94 (S)-enantiomer. Homology models with concomitant docking studies indicated structural reason observed complementary stereopreference.

Language: Английский

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Artificial Small Molecules as Cofactors and Biomacromolecular Building Blocks in Synthetic Biology: Design, Synthesis, Applications, and Challenges

Molecules, Journal Year: 2023, Volume and Issue: 28(15), P. 5850 - 5850

Published: Aug. 3, 2023

Enzymes are essential catalysts for various chemical reactions in biological systems and often rely on metal ions or cofactors to stabilize their structure perform functions. Improving enzyme performance has always been an important direction of protein engineering. In recent years, artificial small molecules have successfully used The types enzymatic metabolic pathways cells can be expanded by the incorporation these either as building blocks proteins nucleic acids, which greatly promotes development application biotechnology. this review, we summarized research including cluster mimics, coenzyme analogs (mNADs), designer cofactors, non-natural nucleotides (XNAs), amino acids (nnAAs), focusing design, synthesis, applications well current challenges synthetic biology.

Language: Английский

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Enantiodivergent Synthesis of Halohydrins by Engineering P450DA Monooxygenases

ACS Catalysis, Journal Year: 2023, Volume and Issue: 13(24), P. 15948 - 15955

Published: Nov. 28, 2023

Chiral γ-halohydrins and β-haloallyl alcohols are important building blocks for the synthesis of pharmacologically active compounds. Direct enantioselective C–H bond hydroxylation halohydrocarbons is an appealing method these Herein, P450DA mutants, which could improve or reverse enantioselectivity, were generated by structure-guided directed evolution based on X-ray crystal structure P450DA-M3. It catalyzed benzylic allylic with regio-, chemo-, enantioselectivity provided desirable enantiomers both chiral (43–94% ee) (79–96% ee), while halogen atoms C═C bonds in molecule remained unreacted. This enzymatic platform represents example catalytic systems achieving enantiodivergent control via protein engineering.

Language: Английский

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Biocatalytic hydroxylation tertiary C-H bonds for synthesis of chiral tertiary alcohols by cytochrome P450

Molecular Catalysis, Journal Year: 2023, Volume and Issue: 553, P. 113791 - 113791

Published: Dec. 23, 2023

Language: Английский

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Diverse N‐Oxidation of Primary Aromatic Amines Controlled by Engineered P450 Peroxizyme Variants Facilitated by Dual‐Functional Small Molecule

Advanced Science, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 16, 2024

Abstract Amine oxidation is an important organic reaction for the production of high‐value N ‐containing compounds. However, it still challenging to control reactivity active ‐centered radicals selectively access ‐oxidation products. Herein, this study reports engineering cytochrome P450BM3 into multifunctional ‐oxidizing enzymes with assistance dual‐functional small molecules (DFSM) produce ‐oxygenation (i.e., p ‐nitrosobenzene, ‐nitrobenzene, and azoxybenzene) one‐electron products oligomeric quinones azobenzene) from aromatic amines. The best mutant, F87A/T268V/V78T/A82T, exclusively gives ‐nitrosobenzene (up 98% selectivity), whereas selectivity ‐nitrobenzene >99% using mutant F87A/T268V/A82T/I263L. Crystal structure analysis reveals that key mutations DFSM exert synergistic effects on catalytic promiscuity by controlling substrate orientation in center. This highlights potential DFSM‐facilitated P450 peroxygenase peroxidase synthesis compounds via controllable amines, substantially expanding chemical space enzymes.

Language: Английский

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C(sp3)-H hydroxylation of Broad-Spectrum Alkanes Catalyzed by an Artificial P450 Peroxygenase Driven by ω-Pyridyl Fatty Acyl Amino Acids

Molecular Catalysis, Journal Year: 2023, Volume and Issue: 550, P. 113618 - 113618

Published: Oct. 13, 2023

Language: Английский

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Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: March 6, 2024

Abstract Regioselective and enantioselective hydroxylation of propargylic C-H bonds are useful reactions but often lack appropriate catalysts. Here a green efficient asymmetric primary secondary C–H at positions has been established. A series optically active alcohols were prepared with high regio- enantioselectivity (up to 99% ee ) under mild reaction conditionsby using P450tol, while the C≡C in molecule remained unreacted. This protocol provides practical method for constructing enantiomerically chiral alcohols. In addition, we also demonstrated that biohydroxylation strategy was able scaled up 2.25 mmol scale production propargyl alcohol 2a yield 196 mg 96% , which’s an important synthetic intermediate antifungal drug Ravuconazole.

Language: Английский

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