Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes

ChemBioChem, Год журнала: 2024, Номер 25(10)

Опубликована: Март 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.

Язык: Английский

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Enzymatic Fluoroethylation by a Fluoroethyl Selenium Analogue of S-Adenosylmethionine

ACS Catalysis, Год журнала: 2024, Номер 14(8), С. 6211 - 6216

Опубликована: Апрель 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.

Язык: Английский

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Engineered Methionine Adenosyltransferase Cascades for Metabolic Labeling of Individual DNA Methylomes in Live Cells

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(27), С. 18722 - 18729

Опубликована: Июнь 29, 2024

Methylation, a widely occurring natural modification serving diverse regulatory and structural functions, is carried out by myriad of S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). The AdoMet cofactor produced from l-methionine (Met) ATP family multimeric methionine adenosyltransferases (MAT). To advance mechanistic functional studies, strategies for repurposing the MAT MTase reactions to accept extended versions transferable group corresponding precursors have been exploited. Here, we used structure-guided engineering mouse MAT2A enable biocatalytic production an analogue, Ado-6-azide, synthetic S-(6-azidohex-2-ynyl)-l-homocysteine (N3-Met). Three engineered variants showed catalytic proficiency with analogues supported DNA derivatization in cascade M.TaqI variant DNMT1 both absence presence competing Met. We then installed two as MAT2A-DNMT1 cascades embryonic stem cells using CRISPR-Cas genome editing. resulting cell lines maintained normal viability methylation levels Dnmt1-dependent azide tags upon exposure N3-Met physiological This first time demonstrates genetically stable system biosynthetic which enables mild metabolic labeling DNMT-specific methylome live mammalian cells.

Язык: Английский

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Engineering of Halide Methyltransferase BxHMT through Dynamic Cross‐Correlation Network Analysis

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(25)

Опубликована: Апрель 16, 2024

Halide methyltransferases (HMTs) provide an effective way to regenerate S-adenosyl methionine (SAM) from homocysteine and reactive electrophiles, such as methyl iodide (MeI) toluene sulfonate (MeOTs). As compared with MeI, the cost-effective unnatural substrate MeOTs can be accessed directly cheap abundant alcohols, but shows only limited reactivity in SAM production. In this study, we developed a dynamic cross-correlation network analysis (DCCNA) strategy for quickly identifying hot spots influencing catalytic efficiency of enzyme, applied it evolution HMT Paraburkholderia xenovorans. Finally, optimal mutant, M4 (V55T/C125S/L127T/L129P), exhibited remarkable improvement, specific activity 4.08 U/mg towards MeOTs, representing 82-fold increase wild-type (WT) enzyme. Notably, also demonstrated positive impact on ability other donors. The structural mechanism behind enhanced enzyme was uncovered by molecular dynamics simulations. Our work not contributes promising biocatalyst regeneration SAM, offers efficient engineering.

Язык: Английский

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Organophosphorus S-adenosyl-L-methionine mimetics: synthesis, stability, and substrate properties

Frontiers in Chemistry, Год журнала: 2024, Номер 12

Опубликована: Авг. 1, 2024

S-Adenosyl-l-methionine (SAM)-mediated methylation of biomolecules controls their function and regulates numerous vital intracellular processes. Analogs SAM with a reporter group in place the S-methyl are widely used to study these However, many analogs chemically unstable that largely limits practical application. We have developed new compound, SAM-PH , which contains an H-phosphinic (-P(O)(H)OH) instead carboxylic group. SAM-P H is significantly more stable than SAM, retains functional activity catechol-O-methyltransferase methyltransferase WBSCR27 reactions. The last associated Williams-Beuren syndrome. Rac-SAM-P was synthesized chemically, while (R,S)-SAM-P its were prepared enzymatically either from methionine (Met-PH) or analog S-adenosyl-l-homocysteine (SAH-P ) using adenosyltransferase 2A halide methyltransferases, respectively. SAH-P undergoes glycoside bond cleavage presence methylthioadenosine nucleosidase like natural SAH. Thus, promising tools for investigating methyltransferases incorporating groups into substrates.

Язык: Английский

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Enzymatic Reactions of S-Adenosyl-L-Methionine: Synthesis and Applications

Biochemistry (Moscow), Год журнала: 2025, Номер 90(S1), С. S105 - S134

Опубликована: Фев. 1, 2025

Язык: Английский

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Chimeric cofactors enable methyltransferase-catalyzed prenylation

Chem, Год журнала: 2025, Номер unknown, С. 102538 - 102538

Опубликована: Апрель 1, 2025

Язык: Английский

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Methyltransferases from RiPP pathways: shaping the landscape of natural product chemistry

Beilstein Journal of Organic Chemistry, Год журнала: 2024, Номер 20, С. 1652 - 1670

Опубликована: Июль 18, 2024

This review article aims to highlight the role of methyltransferases within context ribosomally synthesised and post-translationally modified peptide (RiPP) natural products. Methyltransferases play a pivotal in biosynthesis diverse products with unique chemical structures bioactivities. They are highly chemo-, regio-, stereoselective allowing methylation at various positions. The different possible acceptor regions peptides described this article. Furthermore, we will discuss potential application these as powerful biocatalytic tools synthesis other bioactive compounds. By providing an overview options available, is intended emphasise RiPP their impact on field product chemistry.

Язык: Английский

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Advances in Direct Fluoroalkylation of Organic Substrates with Partially Fluorinated Alkyl Motivs

ACS Catalysis, Год журнала: 2024, Номер 14(21), С. 15879 - 15907

Опубликована: Окт. 11, 2024

Partially fluorinated alkyl groups other than methyl are increasingly playing crucial roles in the development of drugs with diverse biological activities, thus creating an innovative chemical space within organofluorine chemistry. For studies structure–activity relationships, late-stage modification such or substituents into substrates that bear activity is essential. This perspective will study catalytic protocols for direct introduction partially monofluoroalkylated (−CHFR, –CH2CH2F), difluoroalkylated (−CF2Me, –CH2CF2H), trifluoroalkylated (−CHR(CF3), –CH2CF3, –CH2CH2CF3, –CH(Me)CF3, –C(Me)2CF3), and pentafluoropropylated (−CH2C2F5) onto (hetero)aromatic compounds, double bonds, isonitriles, halides, N, O, S atoms.

Язык: Английский

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Engineering of Halide Methyltransferase BxHMT through Dynamic Cross‐Correlation Network Analysis

Angewandte Chemie, Год журнала: 2024, Номер 136(25)

Опубликована: Апрель 16, 2024

Abstract Halide methyltransferases (HMTs) provide an effective way to regenerate S ‐adenosyl methionine (SAM) from homocysteine and reactive electrophiles, such as methyl iodide (MeI) toluene sulfonate (MeOTs). As compared with MeI, the cost‐effective unnatural substrate MeOTs can be accessed directly cheap abundant alcohols, but shows only limited reactivity in SAM production. In this study, we developed a dynamic cross‐correlation network analysis (DCCNA) strategy for quickly identifying hot spots influencing catalytic efficiency of enzyme, applied it evolution HMT Paraburkholderia xenovorans . Finally, optimal mutant, M4 (V55T/C125S/L127T/L129P), exhibited remarkable improvement, specific activity 4.08 U/mg towards MeOTs, representing 82‐fold increase wild‐type (WT) enzyme. Notably, also demonstrated positive impact on ability other donors. The structural mechanism behind enhanced enzyme was uncovered by molecular dynamics simulations. Our work not contributes promising biocatalyst regeneration SAM, offers efficient engineering.

Язык: Английский

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