Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD+ to NADP+ DOI Open Access
Hemant Kumar, Silke Leimkühler

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(22), P. 16067 - 16067

Published: Nov. 8, 2023

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO2 reduction and serve as nicotinamide cofactor recycling enzymes. More recently, CO2-reducing activity dehydrogenases, especially metal-containing has been further explored for efficient atmospheric capture. Here, we investigate binding site dehydrogenase from Rhodobacter capsulatus its specificity toward NAD+ vs. NADP+ reduction. Starting NAD+-specific wild-type RcFDH, key residues were exchanged enable on basis NAD+-bound cryo-EM structure (PDB-ID: 6TG9). It observed that lysine at position 157 (Lys157) β-subunit enzyme is essential NAD+. RcFDH variants had Glu259 either a positively charged or uncharged amino acid additional with NADP+. The FdsBL279R FdsBK276A also showed Kinetic parameters all determined tested able reduce using NADPH electron donor coupled assay phosphite (PTDH), which regenerates NADPH. This makes suitable applications where it can be other use

Language: Английский

Engineering Electron Transfer Pathway of Cytochrome P450s DOI Creative Commons

Jingting He,

Xin Liu, Chun Li

et al.

Molecules, Journal Year: 2024, Volume and Issue: 29(11), P. 2480 - 2480

Published: May 24, 2024

Cytochrome P450s (P450s), a superfamily of heme-containing enzymes, existed in animals, plants, and microorganisms. can catalyze various regional stereoselective oxidation reactions, which are widely used natural product biosynthesis, drug metabolism, biotechnology. In typical catalytic cycle, use redox proteins or domains to mediate electron transfer from NAD(P)H heme iron. Therefore, the main factors determining efficiency include not only themselves but also their redox-partners pathways. this review, pathway engineering strategies system reviewed four aspects: cofactor regeneration, selection redox-partners, redox-partner engineering, electrochemically photochemically driven transfer.

Language: Английский

Citations

10
Engineering of cofactor preference and catalytic activity of methanol dehydrogenase by growth-coupled directed evolution DOI Creative Commons

Jinxing Yang,

Liwen Fan, Guimin Cheng

et al.

Green Carbon, Journal Year: 2024, Volume and Issue: 2(2), P. 242 - 251

Published: April 5, 2024

Methanol, produced from carbon dioxide, natural gas, and biomass, has drawn increasing attention as a promising green feedstock for biomanufacturing due to its sustainable energy-rich properties. NAD+-dependent methanol dehydrogenase (MDH) catalyzes the oxidation of formaldehyde via NADH generation, providing highly active C1 intermediate reducing power subsequent biosynthesis. However, unsatisfactory catalytic efficiency cofactor bias MDH significantly impede valorization, especially in NADPH-dependent Herein, we employed synthetic NADPH auxotrophic Escherichia coli strains growth-coupled selection platforms directed evolution Bacillus stearothermophilus DSM 2334. or generated by MDH-catalyzed enabled growth auxotrophs, establishing positive correlation between cell rate activity. Using this principle, mutants exhibiting 20-fold improvement (kcat/Km) 90-fold specificity switch NAD+ NADP+ without decrease specific enzyme activity, were efficiently screened random semi-rationally designed libraries. We envision that these will advance valorization auxotrophs serve versatile NAD(P)+-dependent enzymes.

Language: Английский

Citations

7
Designing a microbial factory suited for plant chloroplast-derived enzymes to efficiently and green synthesize natural products: capsanthin and capsorubin as examples DOI
Huibin Chen,

Gary Guo,

Qiaoyue Li

et al.

Metabolic Engineering, Journal Year: 2025, Volume and Issue: 88, P. 215 - 227

Published: Jan. 17, 2025

Language: Английский

Citations

0
Reversible Enzyme-Catalysed NAD+/NADH Electrochemistry DOI Creative Commons
Peter D. Giang, Dimitri Niks, Sheron Hakopian

et al.

Chemical Science, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

The formate dehydrogenase subcomplex FdsBG from Cupriavidus necator has been electrochemically activated to reversibly interconvert NAD + /NADH and provide unique insight its kinetics reactivity.

Language: Английский

Citations

0
Product Distribution of Steady–State and Pulsed Electrochemical Regeneration of 1,4‐NADH and Integration with Enzymatic Reaction DOI Creative Commons
Mohammed Ali Saif Al‐Shaibani, Thaleia Sakoleva, Luka A. Živković

et al.

ChemistryOpen, Journal Year: 2024, Volume and Issue: 13(8)

Published: April 12, 2024

Abstract The direct electrochemical reduction of nicotinamide adenine dinucleotide (NAD + ) results in various products, complicating the regeneration crucial 1,4‐NADH cofactor for enzymatic reactions. Previous research primarily focused on steady–state polarization to examine potential impacts product selectivity. However, this study explores influence dynamic conditions selectivity NAD products by comparing two profiles with steady‐state conditions. Our findings reveal that main including 1,4‐NADH, several dimers, and ADP‐ribose, remained consistent across all A minor by–product, 1,6‐NADH, was also identified. distribution varied depending experimental (steady state vs. dynamic) concentration , higher concentrations overpotentials promoting dimerization. optimal yield achieved under low overpotential concentrations. While enhanced at shorter reaction times, they resulted a significant amount unidentified products. Furthermore, assessed using pulsed enoate reductase (XenB) cyclohexenone reduction.

Language: Английский

Citations

3
Efficient production of 1,6-hexanediol from adipic acid by engineering of carboxylate reductase coupled with genetically modified Escherichia coli DOI

Chen Ma,

Na Jiang,

Feiyi Zhuang

et al.

Chemical Engineering Journal, Journal Year: 2025, Volume and Issue: unknown, P. 160034 - 160034

Published: Jan. 1, 2025

Language: Английский

Citations

0
Construction of A Cofactor Self-Sufficient Enzyme Cascade System Coupled with Microenvironmental Engineering for Efficient Biosynthesis of Tetrahydrofolate and its Derivative of L-5-Methyltetrahydrofolate DOI
Zhiqiang Yan, Ling Qin,

Ruirui Qin

et al.

Published: Jan. 1, 2025

Language: Английский

Citations

0
Construction of a Cofactor Self-Sufficient Enzyme Cascade System Coupled with Microenvironmental Engineering for Efficient Biosynthesis of Tetrahydrofolate and Its Derivative of L-5-Methyltetrahydrofolate DOI Open Access
Zhiqiang Yan, Ling Qin,

Ruirui Qin

et al.

Catalysts, Journal Year: 2025, Volume and Issue: 15(3), P. 235 - 235

Published: Feb. 28, 2025

Tetrahydrofolate (THF), the biologically active form of folate, serves as a crucial carrier one-carbon units essential for synthesizing cellular components such amino acids and purine nucleotides in vivo. It also acts an important precursor production pharmaceuticals, including folinate L-5-methyltetrahydrofolate (L-5-MTHF). In this study, we developed efficient enzyme cascade system tetrahydrofolate from incorporating NADPH recycling, explored its application synthesis L-5-MTHF, derivative tetrahydrofolate. To achieve this, first screened dihydrofolate reductases (DHFRs) various organisms, identifying SmDHFR Serratia marcescens with highest catalytic activity. We then conducted comparative analysis formate dehydrogenases (FDHs) different sources, successfully establishing recycling system. further enhance biocatalytic efficiency, optimized key reaction parameters, temperature, pH, ratio, substrate concentration. address challenge pH mismatch dual-enzyme reactions, employed enzymatic microenvironment regulation strategy. This involved covalently conjugating superfolder green fluorescent protein mutant carrying 30 surface negative charges (−30sfGFP), using SpyCatcher/SpyTag modification resulted 2.16-fold increase production, achieving final yield 4223.4 µM. Finally, extended to establish L-5-MTHF NADH recycling. By methylenetetrahydrofolate reductase (MTHFR), produced 389.8 μM folate formaldehyde. work provides novel pathway biosynthesis highlights potential systems tetrahydrofolate-derived compounds.

Language: Английский

Citations

0
Engineering a newly identified alcohol dehydrogenase from Sphingobium Sp. for efficient utilization of nicotinamide cofactors biomimetics DOI Creative Commons

Yichun Zhu,

Jieyu Zhou,

Xiangyuan Gu

et al.

Bioresources and Bioprocessing, Journal Year: 2025, Volume and Issue: 12(1)

Published: May 5, 2025

Abstract Nicotinamide cofactor biomimetics (NCBs) serve as low-cost alternatives to the expensive NAD(P) + /NAD(P)H, holding significant potential for applications in oxidoreductases. In this study, an alcohol dehydrogenase ( Sp ADH2) from Sphingobium sp. SYK-6 was identified utilization of synthetic NCBs. ADH2 exhibited a catalytic activity 11.55 U/g oxidation syringyl when utilizing para -3-carbamoyl-1-(4-carboxybenzyl)pyridin-1-ium p -BANA ) cofactor. Semi-rational engineering led identification key variants (H43L, A290I, H43L/A290I) with enhanced efficiency and specificity using Compared wild-type, variant H43L/A290I 7-fold increase astonishing 6750-fold improvement ratio. Enzymatic characterization reveals that substrate spectrum could change significantly different totally NCBs (tsNCBs). Furthermore, interaction analysis demonstrates critical roles residues 43 290 anchoring release . This study natural ADH capable NCBs, which has never been reported. Importantly, our results provide valuable candidates biology industrial developments, offer guidance ADHs toward cofactors improved performance.

Language: Английский

Citations

0
Formate Dehydrogenase: Recent Developments for NADH and NADPH Recycling in Biocatalysis DOI Creative Commons
Artur Maier, Lindelo M. Mguni, Anna Ngo

et al.

ChemCatChem, Journal Year: 2024, Volume and Issue: 16(21)

Published: July 25, 2024

Abstract Formate dehydrogenases (FDHs) catalyze the oxidation of formate to CO 2 while reducing NAD(P) + NAD(P)H and are classified into two main classes: metal‐dependent (Mo‐ or W‐containing) metal‐independent FDHs. The latter oxygen‐tolerant relevant as a cofactor regeneration system for various bioprocesses gained more attention due their ability reverse reduction. This review gives an overview FDHs, recent advances made in this field, relevance future applications biocatalysis. includes exploitation novel FDHs which have altered co‐substrate specificity well enzyme engineering approaches improve process stability general performance.

Language: Английский

Citations

3