Engineered enzymes for the synthesis of pharmaceuticals and other high-value products

Nature Synthesis, Journal Year: 2024, Volume and Issue: 3(1), P. 19 - 32

Published: Jan. 4, 2024

Language: Английский

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Learning from Protein Engineering by Deconvolution of Multi‐Mutational Variants

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(36)

Published: June 17, 2024

Abstract This review analyzes a development in biochemistry, enzymology and biotechnology that originally came as surprise. Following the establishment of directed evolution stereoselective enzymes organic chemistry, concept partial or complete deconvolution selective multi‐mutational variants was introduced. Early experiments led to finding mutations can interact cooperatively antagonistically with one another, not just additively. During past decade, this phenomenon shown be general. In some studies, molecular dynamics (MD) quantum mechanics/molecular mechanics (QM/MM) computations were performed order shed light on origin non‐additivity at all stages an evolutionary upward climb. Data used construct unique multi‐dimensional rugged fitness pathway landscapes, which provide mechanistic insights different from traditional landscapes. Along related line, biochemists have long tested result introducing two point enzyme for reasons, followed by comparison respective double mutant so‐called cycles, showed only additive effects, but more recently also uncovered cooperative antagonistic non‐additive effects. We conclude suggestions future work, call unified overall picture epistasis.

Language: Английский

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Protein Engineering of Substrate Specificity toward Nitrilases: Strategies and Challenges

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 10, 2025

Nitrilase is extensively applied across diverse sectors owing to its unique catalytic properties. Nevertheless, in industrial production, nitrilases often face issues such as low efficiency, limited substrate range, suboptimal selectivity, and side reaction products, which have garnered heightened attention. With the widespread recognition that structure of enzymes has a direct impact on their properties, an increasing number researchers are beginning optimize functional characteristics by modifying structures, order meet specific or biotechnology application needs. Particularly artificial intelligence era, innovative computer-aided design enzyme engineering offers remarkable opportunities tailor for production high-value products. In this discussion, we will briefly examine structural mechanism nitrilase. An overview protein strategies preference, regioselectivity stereoselectivity explored combined with some representative examples recently terms specificity enzyme. The future research trends field also prospected.

Language: Английский

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Focused rational iterative site-specific mutagenesis (FRISM): A powerful method for enzyme engineering

Molecular Catalysis, Journal Year: 2023, Volume and Issue: 553, P. 113755 - 113755

Published: Dec. 9, 2023

Language: Английский

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Non-Native Site-Selective Enzyme Catalysis

Chemical Reviews, Journal Year: 2023, Volume and Issue: 123(16), P. 10381 - 10431

Published: July 31, 2023

The ability to site-selectively modify equivalent functional groups in a molecule has the potential streamline syntheses and increase product yields by lowering step counts. Enzymes catalyze site-selective transformations throughout primary secondary metabolism, but leveraging this capability for non-native substrates reactions requires detailed understanding of limitations enzyme catalysis how these bounds can be extended protein engineering. In review, we discuss representative examples involving group manipulation C-H bond functionalization. We include illustrative native catalysis, our focus is on cases often using engineered enzymes. then use enzymes chemoenzymatic target-oriented synthesis conclude with survey tools techniques that could expand scope catalysis.

Language: Английский

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Simplification of Corticosteroids Biosynthetic Pathway by Engineering P450BM3

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(6), P. 4117 - 4129

Published: March 4, 2024

Synthesis of corticosteroids, particularly hydrocortisone, is challenging owing to the complex network requiring pairing cytochrome P450s with P450 reductase (CPR) for achieving regionally selective hydroxylation modifications at multiple sites. Herein, we engineered a self-sufficient P450BM3 (CYP102A1 from Bacillus megaterium) effectively reducing traditionally complex, multienzyme cascade process (three steps and six enzymes) hydrocortisone synthesis progesterone (PG) simplified two-step involving least two enzymes. Driven by computational simulation-guided substrate access channel heme center pocket engineering, series variants were gradually designed ability catalyze C16β, C17α, C21, C17α/21 oxidation PG C11α cortexolone (c). Subsequently, molecular dynamics simulations an oxy-ferrous model revealed that glycine mutations residues are repulsive allow more stable exposure above Fe═O. Finally, developed employed construct efficient Escherichia coli catalytic systems, which further achieved 11α/β-hydrocortisone (f/e) production in one pot 1 g/L molar conversion rate 81 84% (912 955 mg/L), respectively. Thus, this study provides feasible strategies simplifying biosynthetic biocatalysts steroidal pharmaceutical production.

Language: Английский

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Catalyzing the future: recent advances in chemical synthesis using enzymes

Current Opinion in Chemical Biology, Journal Year: 2024, Volume and Issue: 83, P. 102536 - 102536

Published: Oct. 5, 2024

Language: Английский

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

JACS Au, Journal Year: 2024, Volume and Issue: 4(4), P. 1591 - 1604

Published: April 9, 2024

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

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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Recent advances in structure‐based enzyme engineering for functional reconstruction

Biotechnology and Bioengineering, Journal Year: 2023, Volume and Issue: 120(12), P. 3427 - 3445

Published: Aug. 28, 2023

Abstract Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is key structure‐based design, which requires elucidation structure–function relationships. Here, we summarize mutations residues different regions, active center, access tunnels, flexible loops, on fine‐tuning performance enzymes, discuss effects altering local structural environment functions. In addition, keep up with recent progress approaches enzyme engineering, aiming provide some guidance how take advantage information.

Language: Английский

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