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Improving Biocatalytic Properties of an Azoreductase via the N-Terminal Fusion of Formate Dehydrogenase.

ABSTRACT: Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)⁺ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDH_C23S and FDH_{C23SD195QY196H} ) of Candida boidinii in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the N-terminus of the fusion protein. FDH_C23S +AzoRo was the most active construct and was further characterized. Enzymatic activities of FDH_C23S +AzoRo decreased compared to parental enzymes but showed improved substrate scope - accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.

SUBMITTER: Ngo ACR

PROVIDER: S-EPMC9305538 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Fusion of Formate Dehydrogenase and Alanine Dehydrogenase as an Amino Donor Regenerating System Coupled to Transaminases.

Project description:Fusion enzymes are attractive tools for facilitating the assembly of biocatalytic cascades for chemical synthesis. This approach can offer great advantages for cooperative redox cascades that need the constant supply of a donor molecule. In this work, we have developed a self-sufficient bifunctional enzyme that can be coupled to transaminase-catalyzed reactions for the efficient recycling of the amino donor (L-alanine). By genetic fusion of an alanine dehydrogenase (AlaDH) and a formate dehydrogenase (FDH), a redox-complementary system was applied to recycle the amino donor and the cofactor (NADH), respectively. AlaDH and FDH were assembled in both combinations (FDH-AlaDH and AlaDH-FDH), with a 2.5-fold higher enzymatic activity of the latter system. Then, AlaDH-FDH was coupled to two different S-selective transaminases for the synthesis of vanillyl amine (10 mM) reaching up to 99 % conversion in 24 h in both cases. Finally, the multienzyme system was reused for at least 3 consecutive cycles when implemented in dialysis-assisted biotransformations.

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Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity.

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Protein Mass Effects on Formate Dehydrogenase.

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Project description:Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD(+) and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2.

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Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha.

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Oxidation-State-Dependent Binding Properties of the Active Site in a Mo-Containing Formate Dehydrogenase.

Project description:Molybdenum-containing formate dehydrogenase H from Escherichia coli (EcFDH-H) is a powerful model system for studies of the reversible reduction of CO2 to formate. However, the mechanism of FDH catalysis is currently under debate, and whether the primary Mo coordination sphere remains saturated or one of the ligands dissociates to allow direct substrate binding during turnover is disputed. Herein, we describe how oxidation-state-dependent changes at the active site alter its inhibitor binding properties. Using protein film electrochemistry, we show that formate oxidation by EcFDH-H is inhibited strongly and competitively by N3-, OCN-, SCN-, NO2-, and NO3-, whereas CO2 reduction is inhibited only weakly and not competitively. During catalysis, the Mo center cycles between the formal Mo(VI)═S and Mo(IV)-SH states, and by modeling chronoamperometry data recorded at different potentials and substrate and inhibitor concentrations, we demonstrate that both formate oxidation and CO2 reduction are inhibited by selective inhibitor binding to the Mo(VI)═S state. The strong dependence of inhibitor-binding affinity on both Mo oxidation state and inhibitor electron-donor strength indicates that inhibitors (and substrates) bind directly to the Mo center. We propose that inhibitors bind to the Mo following dissociation of a selenocysteine ligand to create a vacant coordination site for catalysis and close by considering the implications of our data for the mechanisms of formate oxidation and CO2 reduction.

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Improving kinetic or thermodynamic stability of an azoreductase by directed evolution.

Project description:Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. In this study, five rounds of mutagenesis/recombination followed by high-throughput screening (≈10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50°C than that exhibited by the homodimeric wild type PpAzoR azoreductase from Pseudomonas putida MET94. The characterization using fluorescence, calorimetry and light scattering shows that 1B6 has a folded state slightly less stable than the wild type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant was therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturbed hydrophobic patches and increased the surface net charge of the protein. Variants 2A1 and 2A1-Y179H with increased thermodynamic stability (10 to 20°C higher melting temperature than wild type) were also examined showing the distinctive nature of mutations that lead to improved structural robustness: these occur in residues that are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state.

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