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    Application of reductive amination by heterologously expressed Thermomicrobium roseum L-alanine dehydrogenase to synthesize L-alanine derivatives
    (Elsevier Science Inc, 2023) Dedeakayogullari, Huri; Valjakka, Jarkko; Turunen, Ossi; Yilmazer, Berin; Demir, Garip; Janis, Janne; Binay, Baris
    Unnatural amino acids are unique building blocks in modern medicinal chemistry as they contain an amino and a carboxylic acid functional group, and a variable side chain. Synthesis of pure unnatural amino acids can be made through chemical modification of natural amino acids or by employing enzymes that can lead to novel molecules used in the manufacture of various pharmaceuticals. The NAD+ -dependent alanine dehydrogenase (AlaDH) enzyme catalyzes the conversion of pyruvate to L-alanine by transferring ammonium in a reversible reductive amination activity. Although AlaDH enzymes have been widely studied in terms of oxidative deamination activity, reductive amination activity studies have been limited to the use of pyruvate as a substrate. The reductive amination potential of heterologously expressed, highly pure Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) was examined with regard to pyruvate, a-ketobutyrate, a-ketovalerate and a-ketocaproate. The biochemical properties were studied, which included the effects of 11 metal ions on enzymatic activity for both reactions. The enzyme accepted both derivatives of L-alanine (in oxidative deamination) and pyruvate (in reductive amination) as substrates. While the kinetic KM values associated with the pyruvate derivatives were similar to pyruvate values, the kinetic k(cat) values were significantly affected by the side chain increase. In contrast, K-M values associated with the derivatives of L-alanine (L-a-aminobutyrate, L-norvaline, and L-norleucine) were approximately two orders of magnitude greater, which would indicate that they bind very poorly in a reactive way to the active site. The modeled enzyme structure revealed differences in the molecular orientation between L-alanine/pyruvate and L-norleucine/a-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.
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    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.