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  • Küçük Resim
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    Conserved amino acid residues that affect structural stability of candida boidinii formate dehydrogenase
    (Springer, 2021) Bulut, Huri; Yüksel, Büşra; Gül, Mehmet; Eren, Meryem; Karataş, Ersin; Kara, Nazlı; Yılmazer, Berin; Koçyiğit, Abdurrahim; Labrou, Nikolaos E.; Bi?nay, Barış
    The NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme’s thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73 °C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25 °C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies.
  • Küçük Resim
    Öğe
    Effect of metal Ions on the activity of ten NAD-dependent formate dehydrogenases
    (2020) Bulut, Huri; Valjakka, Jarkko; Yüksel, Büşra; Yılmazer, Berin; Turunen, Ossi; Binay, Barış
    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.