Learning from Protein Engineering by Deconvolution of Multi‐Mutational Variants

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(36)

Опубликована: Июнь 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.

Язык: Английский

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Enzymes in “Green” Synthetic Chemistry: Laccase and Lipase

Molecules, Год журнала: 2024, Номер 29(5), С. 989 - 989

Опубликована: Фев. 24, 2024

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes alternatives to many common catalysts. Their essential advantages high catalytic efficiency, substrate specificity, minimal formation of byproducts, low energy demand. All these benefits make enzymes highly desirable targets academic research industrial development. This review has the modest aim briefly overviewing classification, mechanism action, basic kinetics reaction condition effects that across all six enzyme classes. Special attention is devoted immobilization strategies main tools improve resistance environmental stress factors (temperature, pH solvents) prolong lifecycle biocatalysts. The drawbacks methods such macromolecular crosslinking, solid scaffold carriers, entrapment, surface modification (covalent physical) discussed illustrated using examples. Among hundreds possibly thousands known recently discovered enzymes, hydrolases oxidoreductases distinguished by their relative availability, stability, wide use synthetic applications, which include pharmaceutics, food beverage treatments, clean-up, polymerizations. Two representatives those groups-laccase (an oxidoreductase) lipase (a hydrolase)-are at length, including structure, mechanism, diverse usage. Objective representation current status emerging trends provided conclusions.

Язык: Английский

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Enzymatic Stereodivergent Access to Fluorinated β-Lactam Pharmacophores via Triple-Parameter Engineered Ketoreductases

ACS Catalysis, Год журнала: 2024, Номер 14(8), С. 6358 - 6368

Опубликована: Апрель 11, 2024

Enzyme-catalyzed stereodivergent synthesis to access all possible stereoisomers of organofluorine compounds bearing multiple stereogenic centers remains an important and challenging subject. By integrative data-driven mining mechanism-guided engineering ketoreductases, we identified a biocatalytic platform produce four stereoisomeric fluoroalkyl amino acid esters two vicinal stereocenters. Fast triple-parameter coevolution via semirational CAST/ISM strategy provided the quadruple mutant M5 (A140K/L203T/G92A/V84I) ketoreductase BgADH not only displayed high stereoselectivity toward target (99:1 dr, 99% ee) but also observed with enhanced activity (kcat/Km, 6.3 folds) improved thermostability (T5015, 4 °C). Crystal structural analysis molecular dynamics (MD) simulation studies unveil residues (A140 F148) be key sites that are responsible for control stereoselectivity. The L203T/G92A mutation by affecting conformational distribution α-helix within active-site region, V84I thermal stability strengthening hydrogen bonding network neighboring residues. synthetic utility was further demonstrated substrate scope expansion, gram-scale reactions (648 g L–1 day–1), transformations chiral fluorinated β-lactams antibiotic carbapenem cores.

Язык: Английский

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Synthesis of protein-based catalyst for multi-industrial applications by using agriculture and industrial waste

Polymer Bulletin, Год журнала: 2024, Номер 81(14), С. 12633 - 12653

Опубликована: Май 19, 2024

Язык: Английский

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Complementary Distant and Active Site Mutations Simultaneously Enhance Catalytic Activity and Thermostability of α-Galactosidase

Journal of Agricultural and Food Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Фев. 3, 2025

The industrial applications of enzymes are limited due to the activity-stability trade-off, which implies that improvement thermostability often accompanies decreased activity. This study presents a dual-strategy approach simultaneously improve catalytic efficiency and α-galactosidase galV from Anoxybacillus vitaminiphilus WMF1. Our integrated method combines computational analysis with enzyme property prediction selectively target modify region residues distant active site. We identified experimentally validated mutations activity without compromising stability further increased through additional distant-site mutations. resulting mutant variant N549Q/T550N/Y634F demonstrated 6.2-fold increase in 3.2-fold half-life at 65 °C. Molecular dynamics (MD) simulations supported structural basis for observed enhancements. offers refined strategy engineering α-galactosidases improved applicability, overcoming traditional trade-offs between stability. Hydrolytic toward raffinose family oligosaccharides (RFOs) was using soymilk as model substrate, demonstrating significant practical potential.

Язык: Английский

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Extensive gene mining and facile engineering of a novel carbonyl reductase for asymmetric synthesis of anti-aging (S)-Pro-Xylane from d-xylose

International Journal of Biological Macromolecules, Год журнала: 2025, Номер unknown, С. 140976 - 140976

Опубликована: Фев. 1, 2025

Язык: Английский

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Rational Design of Spontaneous Self-Cyclization Enzymes In Vivo and In Vitro with Improved Thermal Tolerance and Activity

ACS Catalysis, Год журнала: 2024, Номер 14(7), С. 5469 - 5480

Опубликована: Март 28, 2024

Enzymes have selectivity, require mild catalytic conditions, and are important cornerstones in many industrial processes. Protein self-cyclization has opened up the possibility of preserving fragile enzymes during long-term high-temperature catalysis. However, mechanism for improvement thermal tolerance not been elucidated, severely limiting their applications. Herein, we provide a strategy rational design fusion proteins based on structural analysis to obtain cyclized with improved properties. First, constructed that preferentially translated SpyCatcher (CBT) or SpyTag (TBC), both which could form stable single significantly tolerance. Especially, half-life TBC obtained by modifying N-terminal at 40 °C was 10.83 times wild enzymes. Structural revealed terminus protein, SpyCatcher, folded into conformation adversely affected stability. In addition, structure pocket orientation residues CBT were different from those wild-type enzymes, led decrease activity. These conclusions confirmed when another enzyme, sucrose phosphorylase, cyclized. Finally, glucosidase also superior strain preparation ginsenoside F1 high titers as whole-cell catalyst extended use. conclusion, demonstrated first time conjugated long oligopeptide activity stability It necessary translate units less steric hindrance reduce impact protein structure. The provides simple effective modification poor tolerance, providing considerable prospects biosynthesis vivo vitro.

Язык: Английский

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Modular Access to Chiral Amines via Imine Reductase-Based Photoenzymatic Catalysis

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(20), С. 14278 - 14286

Опубликована: Май 10, 2024

The development of catalysts serves as the cornerstone innovation in synthesis, exemplified by recent discovery photoenzymes. However, repertoire naturally occurring enzymes repurposed direct light excitation to catalyze new-to-nature photobiotransformations is currently limited flavoproteins and keto-reductases. Herein, we shed on imine reductases (IREDs) that remote C(sp3)–C(sp3) bond formation, providing a previously elusive radical hydroalkylation enamides for accessing chiral amines (45 examples with up 99% enantiomeric excess). Beyond their natural function catalyzing two-electron reductive amination reactions, upon visible-light or synergy synthetic photoredox catalyst, IREDs are tune non-natural photoinduced single-electron processes. By conducting wet mechanistic experiments computational simulations, unravel how engineered intermediates toward productive enantioselective pathway. This work represents promising paradigm harnessing nature's asymmetric transformations remain challenging through traditional chemocatalytic methods.

Язык: Английский

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Fermentative Production of Ergothioneine by Exploring Novel Biosynthetic Pathway and Remodulating Precursor Synthesis Pathways

Journal of Agricultural and Food Chemistry, Год журнала: 2024, Номер 72(25), С. 14264 - 14273

Опубликована: Июнь 11, 2024

Ergothioneine (EGT) is a naturally occurring derivative of histidine with diverse applications in the medicine, cosmetic, and food industries. Nevertheless, its sustainable biosynthesis faces hurdles due to limited biosynthetic pathways, complex metabolic network precursors, high cost associated fermentation. Herein, efforts were made address these limitations first by reconstructing novel EGT pathway from

Язык: Английский

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Atroposelective Synthesis of Axial Biaryls by Dynamic Kinetic Resolution Using Engineered Imine Reductases

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(41)

Опубликована: Июль 17, 2024

Axially chiral biaryl compounds are ubiquitous scaffolds in natural products, bioactive molecules, ligands and catalysts, but biocatalytic methods for their asymmetric synthesis limited. Herein, we report a highly efficient route the atroposelective of biaryls by dynamic kinetic resolution (DKR). This DKR approach features transient six-membered aza-acetal-bridge-promoted racemization followed an imine reductase (IRED)-catalyzed stereoselective reduction to construct axial chirality under ambient conditions. Directed evolution IRED from Streptomyces sp. GF3546 provided variant (S-IRED-Ss-M11) capable catalyzing process access variety aminoalcohols high yields excellent enantioselectivities (up 98 % yield >99 : 1 enantiomeric ratio). Molecular dynamics simulation studies on S-IRED-Ss-M11 revealed origin its improved activity atroposelectivity. By exploiting substrate promiscuity IREDs power directed evolution, our work further extends biocatalysts' toolbox challenging axially molecules.

Язык: Английский

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