Opportunities and challenges in design and optimization of protein function

Nature Reviews Molecular Cell Biology, Journal Year: 2024, Volume and Issue: 25(8), P. 639 - 653

Published: April 2, 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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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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Aminodealkenylation: Ozonolysis and copper catalysis convert C(sp ³ )–C(sp ² ) bonds to C(sp ³ )–N bonds

Science, Journal Year: 2023, Volume and Issue: 381(6660), P. 877 - 886

Published: Aug. 24, 2023

Great efforts have been directed toward alkene π bond amination. In contrast, analogous functionalization of the adjacent C(sp

Language: Английский

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Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes

ChemBioChem, Journal Year: 2024, Volume and Issue: 25(10)

Published: March 13, 2024

Abstract S ‐Adenosyl‐ l ‐methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand chemistry accessible with SAM‐dependent transferases and go beyond methylation Here we present efficient enzyme engineering strategy to synthesize different from “off‐the‐shelf” iodoalkanes through enzymatic alkylation ‐adenosyl‐ ‐homocysteine (SAH). This was achieved by mutating multiple hydrophobic structurally dynamic amino acids simultaneously. Combinatorial mutagenesis guided natural acid diversity generated a highly functional mutant library. approach increased speed as well scale providing panel optimized enzymes orders magnitude higher activities substrates just one round engineering. The exhibit catalytic efficiencies up 31 M −1 s , convert iodoalkanes, bearing cyclopropyl or aromatic moieties, catalyze ‐alkylation SAH very high stereoselectivities (>99 % de ). We further report throughput chromatographic screening system reliable rapid analog analysis. believe that described herein will advance field biocatalytic enabling regeneration reagents.

Language: Английский

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Enzymatic Fluoroethylation by a Fluoroethyl Selenium Analogue of S-Adenosylmethionine

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(8), P. 6211 - 6216

Published: April 9, 2024

Fluorine is a unique element with important roles in medicinal chemistry, agrochemistry, and materials chemistry. The fluoroethyl group an fluoroalkyl functional unit that widely used clinical drugs, 19F probes 18F PET diagnostic drugs. Chemo- regioselective fluoroethylation difficult chemical synthesis. To date, no enzymatic reaction for selective has been reported. Based on the widespread natural methyl donor S-adenosine-l-methionine (SAM), we designed synthesized SAM analogue (FEt-SAM). A stability study revealed FEt-SAM was very labile under physiological conditions gave fluorine-elimination product vinyl-SAM. We circumvented this problem by replacing S Se to give Se-adenosyl-l-selenomethionine (FEt-SeAM). By using halide methyltransferase (HMT) its mutant situ production of FEt-SeAM, created cascade reactions HMT methyltransferases fluoroethylated several O-, N-, S-, C-nucleophiles. For did not recognize FEt-SeAM well, such as DnrK NovO, simple mutagenesis conserved hydrophobic residues (Leu Ile) binding pocket smaller amino acids significantly increased activities. Therefore, have provided useful tool late-stage products This method could also be enzymatically prepare NMR tests.

Language: Английский

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A New Age of Biocatalysis Enabled by Generic Activation Modes

JACS Au, Journal Year: 2024, Volume and Issue: 4(6), P. 2068 - 2080

Published: May 31, 2024

Biocatalysis is currently undergoing a profound transformation. The field moves from relying on nature's chemical logic to discipline that exploits generic activation modes, allowing for novel biocatalytic reactions and, in many instances, entirely new chemistry. Generic modes enable wide range of reaction types and played pivotal role advancing the fields organo- photocatalysis. This perspective aims summarize principal harnessed enzymes develop biocatalysts. Although extensively researched past, highlighted when applied within enzyme active sites, facilitate transformations have largely eluded efficient selective catalysis. advance attributed multiple tunable interactions substrate binding pocket precisely control competing pathways transition states. We will highlight cases synthetic methodologies achieved by engineered provide insights into potential future developments this rapidly evolving field.

Language: Английский

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SubTuner leverages physics-based modeling to complement AI in enzyme engineering toward non-native substrates

Chem Catalysis, Journal Year: 2025, Volume and Issue: unknown, P. 101334 - 101334

Published: March 1, 2025

Language: Английский

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Addressing epistasis in the design of protein function

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(34)

Published: Aug. 12, 2024

Mutations in protein active sites can dramatically improve function. The site, however, is densely packed and extremely sensitive to mutations. Therefore, some mutations may only be tolerated combination with others a phenomenon known as epistasis. Epistasis reduces the likelihood of obtaining improved functional variants slows natural lab evolutionary processes. Research has shed light on molecular origins epistasis its role shaping trajectories outcomes. In addition, sequence- AI-based strategies that infer epistatic relationships from mutational patterns or experimental evolution data have been used design variants. recent years, combinations such approaches atomistic calculations successfully predicted highly combinatorial sites. These were thousands active-site variants, demonstrating that, while our understanding remains incomplete, determinants are critical for accurate now sufficiently understood. We conclude space explored by expanded enhance activities discover new ones. Furthermore, opens way systematically exploring sequence structure impacts function, deepening control over activity.

Language: Английский

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Selective Biocatalytic N‐Methylation of Unsaturated Heterocycles

Angewandte Chemie International Edition, Journal Year: 2022, Volume and Issue: 61(48)

Published: Oct. 7, 2022

Methods for regioselective N-methylation and -alkylation of unsaturated heterocycles with "off the shelf" reagents are highly sought-after. This reaction could drastically simplify synthesis privileged bioactive molecules. Here we report engineered natural methyltransferases challenging N-(m)ethylation heterocycles, including benzimidazoles, benzotriazoles, imidazoles indazoles. The reactions performed through a cyclic enzyme cascade that consists two using only iodoalkanes or methyl tosylate as simple reagents. method enables selective important molecules otherwise difficult to access, proceeds high regioselectivity (r.r. up >99 %), yield (up 99 on preparative scale, nearly equimolar concentrations starting materials.

Language: Английский

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