10 results on '"Ruupunen, Jouni"'
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2. Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 −) to formate
- Author
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Aslan, Aşkn Sevinç, Valjakka, Jarkko, Ruupunen, Jouni, Yildirim, Deniz, Turner, Nicholas J., Turunen, Ossi, and Binay, Barş
- Published
- 2017
- Full Text
- View/download PDF
3. Engineered Formate Dehydrogenase Fromchaetomium Thermophilum, A Promising Enzymatic Solution For Biotechnical Co(2)Fixation
- Author
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Cakar, Mehmet M., Ruupunen, Jouni, Mangas-Sanchez, Juan, Birmingham, William R., Yildirim, Deniz, Turunen, Ossi, Turner, Nicholas J., Valjakka, Jarkko, and Binay, Baris
- Abstract
Objectives Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH fromChaetomium thermophilum(CtFDH) with enhanced reduction activity in the conversion of CO(2)into formic acid. Results Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO(2(g))and HCO3-. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (k(cat)/K-M) compared to that of the wild-type for HCO(3)(-)reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase fromStreptomyces coelicolorA3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-typeCtFDH. Conclusions Two variants presenting up to five-fold increase in catalytic efficiency andk(cat)were obtained and characterised. They constitute a promising enzymatic alternative for CO(2)utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.
- Published
- 2020
4. Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO2 fixation.
- Author
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Çakar, Mehmet M., Ruupunen, Jouni, Mangas-Sanchez, Juan, Birmingham, William R., Yildirim, Deniz, Turunen, Ossi, Turner, Nicholas J., Valjakka, Jarkko, and Binay, Barış
- Subjects
CHAETOMIUM ,ALCOHOL dehydrogenase ,STREPTOMYCES coelicolor ,FORMIC acid ,BIOCATALYSIS ,DEHYDROGENASES ,COFACTORS (Biochemistry) - Abstract
Objectives: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO
2 . The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. Results: Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3 − . The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat /KM ) compared to that of the wild-type for HCO3 − reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. Conclusions: Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements. [ABSTRACT FROM AUTHOR]- Published
- 2020
- Full Text
- View/download PDF
5. Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3-) to formate
- Author
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Aslan, Askin Sevinc, Valjakka, Jarkko, Ruupunen, Jouni, Yildirim, Deniz, Turner, Nicholas J., Turunen, Ossi, Binay, Baris, and Çukurova Üniversitesi
- Subjects
molecular modeling ,kinetic parameters with hydrogen carbonate ,catalytic mechanism ,biotransformation of CO2 ,NAD(+)-dependent formate dehydrogenase - Abstract
WOS: 000397129400007 PubMed ID: 27887026 While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO-). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3-) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO(3)w(-) as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3- to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO3- reduced the reaction rate. CtFDH was modeled in the presence of HCO3- showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3-, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions. TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [214Z292] This work was supported by a grant from the TUBITAK (Grant number: 214Z292).
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- 2017
6. Functional effects of active site mutations in NAD+-dependent formate dehydrogenases on transformation of hydrogen carbonate to formate
- Author
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Pala, Uğur, primary, Yelmazer, Berin, additional, Çorbacıoğlu, Meltem, additional, Ruupunen, Jouni, additional, Valjakka, Jarkko, additional, Turunen, Ossi, additional, and Binay, Barış, additional
- Published
- 2018
- Full Text
- View/download PDF
7. Catalytic mechanism for reduction of bicarbonate in the active site of formate dehydrogenase
- Author
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Ruupunen, Jouni, BioMediTech - BioMediTech, and University of Tampere
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Bioteknologian tutkinto-ohjelma - Degree Programme in Biotechnology - Abstract
Formate dehydrogenases (FDHs) are a set of mostly homodimeric oxidoreductases that catalyze the oxidation of formate to carbon dioxide with the concurrent reduction of NAD+ to NADH. These enzymes are common to methylotropic yeasts and bacteria in which they are vital in the catabolism of methanol and methane. FDHs catalyze relatively simple hydride transfer reaction in which formate donates a hydride to the C4N atom of the nicotinamide ring of NAD+, yielding carbon dioxide and NADH. During the recent decades, FDHs have been utilized in coenzyme regeneration systems that produce NADH which would be expensive to manufacture by conventional chemical synthesis. Another promising application of FDHs is carbon fixation from the atmosphere and the oceans using the reverse reaction, the reduction of carbon dioxide to formate. Recent experimental evidence suggests that formate dehydrogenases are able to reduce bicarbonate to formate as well. The aim of this thesis was to develop a model for the catalytic mechanism for the reduction of bicarbonate in the active site of FDH from bacterium Pseudomonas sp. strain 101. For this purpose, three different initial models for classical molecular dynamics simulations were constructed. The PDB structure 2NAD was used as a basis for all models. In addition to the main simulation which included bicarbonate and NADH in the active site, two reference simulations were performed as well. In the first one, formate and NAD+ were simulated in the active site to ensure that the results would be comparable to earlier computational studies on the same subject. The purpose of the second reference simulation was to observe if carbon dioxide could react to formate in the presence of NADH. It was discovered that bicarbonate settled in a conformation ideal for hydride transfer during the simulation. However, the process itself was a time-consuming process, taking up to 3 nanoseconds of simulation time. Experimental results seemed to support this observation. Based on the optimal conformation of bicarbonate, a model for the catalytic mechanism was developed. In the model, the hydride donated by NADH would form a covalent bond to the carbon atom of bicarbonate, resulting in a surplus of electrons within the molecule. This would cause the complex formed by bicarbonate and the hydride to break into formate and hydroxide ions. In the reference simulation with formate the optimal conformations for hydride transfer were not observed, but this was probably due to the inaccuracies of the classical molecular dynamics method described in earlier studies. Carbon dioxide seemed to have a destabilizing effect on the active site. The reverse reaction could still be possible as carbon dioxide occasionally drifted close to the hydride of NADH bound to the C4N.
- Published
- 2016
8. Functional effects of active site mutations in NAD + -dependent formate dehydrogenases on transformation of hydrogen carbonate to formate.
- Author
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Pala, Uğur, Yelmazer, Berin, Çorbacıoğlu, Meltem, Ruupunen, Jouni, Valjakka, Jarkko, Turunen, Ossi, and Binay, Barış
- Subjects
CANDIDA ,BACTERIAL mutation ,BINDING sites ,NAD (Coenzyme) ,FORMATES ,DEHYDROGENASES ,CARBONATES - Abstract
Conversion of hydrogen carbonate to formate by mutants of Candida methylica (Cm FDH) and Chaetomium thermophilum (Ct FDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In Ct FDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduction) as a function of substrate concentration. The mutation Asn120Cys in Ct FDH increased 6.5-fold the K
M , indicating that substrate binding was weakened. A 6.5-fold increase of kcat compensated the lower affinity suggesting that product release was improved. The corresponding mutation Asn119Cys in Cm FDH inactivated the enzyme for both substrates. Molecular dynamics simulations indicated that the active site dimensions change differently with different substrates after mutations, and in the mutant Asn120Cys of Ct FDH, hydrogen carbonate adopted better reactive position than formate. With hydrogen carbonate, the active site enlarged enough for two hydrogen carbonate molecules to be placed there. The change of Asn119 to bulky Tyr or His in Cm FDH requires changes in the active site to accommodate the substrate; activity with formate was retained but not with hydrogen carbonate. This study showed that the active site of FDHs can be modified radically, which gives possibilities for further enzyme engineering to improve the reaction with hydrogen carbonate or carbon dioxide for enzymatic fixing of carbon dioxide. [ABSTRACT FROM AUTHOR]- Published
- 2018
- Full Text
- View/download PDF
9. Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3-)to formate.
- Author
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Aslan, Aşkın Sevinç, Valjakka, Jarkko, Ruupunen, Jouni, Yildirim, Deniz, Turner, Nicholas J., Turunen, Ossi, and Binay, Barış
- Subjects
CHAETOMIUM ,DEHYDROGENASES ,CHEMICAL synthesis ,FORMATES ,BIOENGINEERING - Abstract
While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO
2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO- ). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3 - ) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3 - as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3 - to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO3 - reduced the reaction rate. CtFDH was modeled in the presence of HCOHCO3 - showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3 - , resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
10. Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 -) to formate.
- Author
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Aslan AS, Valjakka J, Ruupunen J, Yildirim D, Turner NJ, Turunen O, and Binay B
- Subjects
- Biotransformation, Catalytic Domain, Formate Dehydrogenases chemistry, Formate Dehydrogenases genetics, Kinetics, Models, Molecular, Oxidation-Reduction, Protein Engineering, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Bicarbonates metabolism, Chaetomium enzymology, Formate Dehydrogenases metabolism, Formates metabolism
- Abstract
While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO
2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO- ). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3 - ) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3 - as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3 - to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO3 - reduced the reaction rate. CtFDH was modeled in the presence of HCO3 - showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3 - , resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)- Published
- 2017
- Full Text
- View/download PDF
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