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Effect of Metal Ions on the Activity of Ten NAD-Dependent Formate Dehydrogenases

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Abstract

NAD-dependent formate dehydrogenase (FDH) enzymes are frequently used in industrial and scientific applications. FDH is a reversible enzyme that reduces the NAD molecule to NADH and produces CO2 by oxidation of the formate ion, whereas it causes CO2 reduction in the reverse reaction. Some transition metal elements – Fe3+, Mo6+ and W6 + – can be found in the FDH structure of anaerobic and archaeal microorganisms, and these enzymes require cations and other redox-active cofactors for their FDH activity. While NAD-dependent FDHs do not necessarily require any metal cations, the presence of various metal cations can still affect FDH activities. To study the effect of 11 different metal ions, NAD-dependent FDH enzymes from ten different microorganisms were tested: Ancylobacter aquaticus (AaFDH), Candida boidinii (CboFDH), Candida methylica (CmFDH), Ceriporiopsis subvermispora (CsFDH), Chaetomium thermophilum (CtFDH), Moraxella sp. (MsFDH), Myceliophthora thermophila (MtFDH), Paracoccus sp. (PsFDH), Saccharomyces cerevisiae (ScFDH) and Thiobacillus sp. (TsFDH). It was found that metal ions (mainly Cu2+ and Zn2+) could have quite strong inhibition effects on several enzymes in the forward reaction, whereas several cations (Li+, Mg2+, Mn2+, Fe3+ and W6+) could increase the forward reaction of two FDHs. The highest activity increase (1.97 fold) was caused by Fe3+ in AaFDH. The effect on the reverse reaction was minimal. The modelled structures of ten FDHs showed that the active site is formed by 15 highly conserved amino acid residues spatially settling around the formate binding site in a conserved way. However, the residue differences at some of the sites close to the substrate do not explain the activity differences. The active site space is very tight, excluding water molecules, as observed in earlier studies. Structural examination indicated that smaller metal ions might be spaced close to the active site to affect the reaction. Metal ion size showed partial correlation to the effect on inhibition or activation. Affinity of the substrate may also affect the sensitivity to the metal’s effect. In addition, amino acid differences on the protein surface may also be important for the metal ion effect.

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Abbreviations

AaFDH:

Ancylobacter aquaticus FDH

CboFDH:

Candida boidinii FDH

CmFDH:

Candida methylica FDH

CsFDH:

Ceriporiopsis subvermispora FDH

CtFDH:

Chaetomium thermophilum FDH

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

KDa:

Kilodalton

mM:

Millimolar

MsFDH:

Moraxella sp. FDH

MtFDH:

Myceliopthora thermophila FDH

NAD:

Nicotinamide adenine dinucleotide

NADH:

Nicotinamide adenine dinucleotide hydrogen

PsFDH:

Paracoccus sp. FDH

SceFDH:

Saccharomyces cerevisiae FDH

TsFDH:

Thiobacillus sp. FDH

μM:

Micromolar

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Acknowledgements

Special thanks to Gebze Technical University Enzyme Test and Consultancy Center for providing infrastructure during the research.

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Authors and Affiliations

  1. Medical Biochemistry Department, Faculty of Medicine, Istinye University, Istanbul, Turkey

    Huri Bulut

  2. Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland

    Jarkko Valjakka

  3. Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey

    Busra Yuksel

  4. Molecular Biology and Genetics Department, Gebze Technical University, Kocaeli, Turkey

    Berin Yilmazer

  5. School of Forest Sciences, Faculty of Science and Forestry, University of Eastern Finland, Joensuu, Finland

    Ossi Turunen

  6. Department of Bioengineering, Gebze Technical University, Kocaeli, Turkey

    Baris Binay

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  1. Huri Bulut
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  2. Jarkko Valjakka
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Correspondence to Baris Binay.

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Bulut, H., Valjakka, J., Yuksel, B. et al. Effect of Metal Ions on the Activity of Ten NAD-Dependent Formate Dehydrogenases. Protein J 39, 519–530 (2020). https://doi.org/10.1007/s10930-020-09924-x

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