Engineering enzyme activity using an expanded amino acid alphabet

Protein Engineering Design and Selection, Journal Year: 2022, Volume and Issue: 36

Published: Nov. 10, 2022

Abstract Enzyme design and engineering strategies are typically constrained by the limited size of nature’s genetic alphabet, comprised only 20 canonical amino acids. In recent years, site-selective incorporation non-canonical acids (ncAAs) via an expanded code has emerged as a powerful means inserting new functional components into proteins, with hundreds structurally diverse ncAAs now available. Here, we highlight how emergence repertoire opened avenues in enzyme engineering. have been used to probe complex biological mechanisms, augment function and, most ambitiously, embed catalytic mechanisms protein active sites that would be challenging access within constraints code. We predict studies reviewed this article, along further advances expansion technology, will establish ncAA increasingly important tool for biocatalysis coming years.

Language: Английский

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Biocatalytic Friedel‐Crafts Reactions

ChemCatChem, Journal Year: 2022, Volume and Issue: 14(18)

Published: July 11, 2022

Abstract Friedel‐Crafts alkylation and acylation reactions are important methodologies in synthetic industrial chemistry for the construction of aryl‐alkyl aryl‐acyl linkages that ubiquitous bioactive molecules. Nature also exploits these many biosynthetic processes. Much work has been done to expand application enzymes unnatural substrates through directed evolution. The promise such biocatalysts is their potential supersede inefficient toxic chemical approaches reactions, with mild operating conditions ‐ hallmark enzymes. Complementary created bio‐hybrid catalysts consisting anchored into biomolecular scaffolds, which display same desirable characteristics. In this Review, we summarise efforts, focussing on both mechanistic aspects considerations, concluding an overview frontiers field routes towards more efficient benign future humankind.

Language: Английский

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Design of Artificial Enzymes: Insights into Protein Scaffolds

ChemBioChem, Journal Year: 2022, Volume and Issue: 24(6)

Published: Nov. 24, 2022

Abstract The design of artificial enzymes has emerged as a promising tool for the generation potent biocatalysts able to promote new‐to‐nature reactions with improved catalytic performances, providing powerful platform wide‐ranging applications and better understanding protein functions structures. selection an appropriate scaffold plays key role in process. This review aims give general overview most common scaffolds that can be exploited enzymes. Several examples are discussed categorized according strategy used biocatalyst, namely functionalization natural enzymes, creation new site bearing wide hydrophobic pocket de novo design. is concluded by comparison these different methods our perspective on topic.

Language: Английский

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Expanding the Genetic Code: Incorporation of Functional Secondary Amines via Stop Codon Suppression

ChemCatChem, Journal Year: 2023, Volume and Issue: 16(1)

Published: Aug. 31, 2023

Abstract Enzymes are attractive catalysts for chemical industries, and their use has become a mature alternative to conventional methods. However, biocatalytic approaches often restricted metabolic less complex reactivities, given the limited amount of functional groups present. This drawback can be addressed by incorporating non‐canonical amino acids (ncAAs) harboring new‐to‐nature groups. Inspired organocatalysis, we report design, synthesis characterization panel ncAAs secondary amines cellular incorporation into different protein scaffolds. D / L ‐pyrrolidine‐ ‐piperidine‐based were successfully site‐specifically incorporated proteins via stop codon suppression methodology. To demonstrate utility these ncAAs, catalytic performance obtained artificial enzymes was investigated in model Michael addition reaction. The pyrrolidine‐ piperidine‐ based significantly expands available toolbox engineering biology applications.

Language: Английский

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Noncanonical Amino Acids: Bringing New-to-Nature Functionalities to Biocatalysis

Chemical Reviews, Journal Year: 2024, Volume and Issue: 124(19), P. 10877 - 10923

Published: Sept. 27, 2024

Biocatalysis has become an important component of modern organic chemistry, presenting efficient and environmentally friendly approach to synthetic transformations. Advances in molecular biology, computational modeling, protein engineering have unlocked the full potential enzymes various industrial applications. However, inherent limitations natural building blocks sparked a revolutionary shift.

Language: Английский

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Evolutionary Specialization of a Promiscuous Designer Enzyme

ACS Catalysis, Journal Year: 2025, Volume and Issue: 15(3), P. 1544 - 1552

Published: Jan. 13, 2025

The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning metabolic networks. It also acts as both curse and blessing engineers, where enzymes that have undergone directed may exhibit exquisite selectivity at expense diminished overall catalytic repertoire. We previously performed two independent campaigns on designer leverages unique properties noncanonical amino acid (ncAA) para-aminophenylalanine (pAF) residue, resulting evolved variants which are specialized. Here, we combine mutagenesis, crystallography, computation reveal molecular basis specialization phenomenon. In one variant, unexpected change quaternary structure biases substrate dynamics promote enantioselective catalysis, while other demonstrates synergistic cooperation between side chains pAF residue form semisynthetic machinery.

Language: Английский

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Light-Driven Deracemization by a Designed Photoenzyme

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: April 12, 2025

The creation of enzymes with abiological abilities offers exciting opportunities to access new-to-nature biocatalysis beyond that found in nature. Here, we repurpose a novel protein scaffold, CTB10, as an artificial photoenzyme through genetic code expansion. It enables catalytic deracemization cyclopropane, process remains inaccessible traditional due its thermodynamically unfavorable Following structural optimization directed evolution, broad substrate scope high enantioselectivities is achieved. Furthermore, the crystal structure CTB10-based photoenzyme-substrate complex well demonstrates how chiral cavity sculpted promote efficient and selective light-enabled deracemization. Therefore, this study unlocks potential for achieving challenging biocatalysis.

Language: Английский

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Enzymatic Synthesis of Saturated Bioisosteres of Ortho‐Substituted Benzenes by Artificial Photoenzyme

Chemistry - A European Journal, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 17, 2025

Saturated bioisosteres of ortho-substituted benzenes are significant interest due to their enhanced pharmacokinetic properties, such as improved metabolic stability and reduced toxicity, making them valuable in drug design development. However, efficient synthesis remains a challenge organic chemistry. Herein, we report the biocatalytic saturated using engineered artificial photoenzymes. The photoenzyme, incorporating genetically encoded unnatural amino acids with benzophenone photosensitizer residue, facilitate formation chiral moderate enantiomeric excess via energy transfer process. Our results demonstrate versatility photoenzymes mediating new-to-nature reactions that difficult achieve conventional chemical or enzymatic methods.

Language: Английский

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A Dual Anchoring Strategy for the Directed Evolution of Improved Artificial Transfer Hydrogenases Based on Carbonic Anhydrase

ACS Central Science, Journal Year: 2021, Volume and Issue: 7(11), P. 1874 - 1884

Published: Nov. 11, 2021

Artificial metalloenzymes result from anchoring a metal cofactor within host protein. Such hybrid catalysts combine the selectivity and specificity of enzymes with versatility (abiotic) transition metals to catalyze new-to-nature reactions in an evolvable scaffold. With aim improving localization arylsulfonamide-bearing iridium-pianostool catalyst human carbonic anhydrase II (hCAII) for enantioselective reduction prochiral imines, we introduced covalent linkage between guest. Herein, show that judiciously positioned cysteine residue reacts p-nitropicolinamide ligand bound iridium afford additional sulfonamide linkage. Three rounds directed evolution, performed on dually anchored cofactor, led improved activity harmaline (up 97% ee (R) >350 turnovers preparative scale). To evaluate substrate scope, best hits each generation were tested eight substrates. X-ray analysis, carried out at various stages evolutionary trajectory, was used scrutinize (i) nature as well (ii) remodeling substrate-binding pocket.

Language: Английский

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Tandem Friedel‐Crafts‐Alkylation‐Enantioselective‐Protonation by Artificial Enzyme Iminium Catalysis

ChemCatChem, Journal Year: 2022, Volume and Issue: 14(8)

Published: Feb. 8, 2022

The incorporation of organocatalysts into protein scaffolds holds the promise overcoming some limitations this powerful catalytic approach. Previously, we showed that non-canonical amino acid para-aminophenylalanine non-enzymatic scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for Friedel-Crafts alkylation indoles with enals. unnatural aniline side-chain is directly involved in catalysis, operating via well-known organocatalytic iminium-based mechanism. In study, show LmrR_pAF can enantioselectively form tertiary carbon centres not only during C-C bond formation, but also by protonation, delivering proton to one face prochiral enamine intermediate. importance various side-chains pocket distinct from reaction without two particularly important residues were probed exhaustive mutagenesis.

Language: Английский

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