Additivity of the Stabilization Effect of Single Amino Acid Substitutions in Triple Mutants of Recombinant Formate Dehydrogenase from the Soybean Glycine max

Алексеева, А. А.; Kargov, I.S.; Kleimenov, S. Yu.; Савин, С. С.; Тишков, В. И.

doi:10.32607/20758251-2015-7-3-55-64

Cited by 5 publications

(2 citation statements)

References 12 publications

(25 reference statements)

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“…FDH from Glycine max was kindly provided by Prof. Tishkov from Lomonosov Moscow State University (Moscow, Russia). The expression and purification procedures can be found in [ 25 ]. Deionized (DI) water (18.2 MΩ·cm Werner Easypure II system, Leverkusen, Germany) was used in all experiments.…”

Section: Methodsmentioning

confidence: 99%

Up- and Down-Regulation of Enzyme Activity in Aggregates with Gold-Covered Magnetic Nanoparticles Triggered by Low-Frequency Magnetic Field

Veselov,

Efremova,

Prusov

et al. 2024

Nanomaterials

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The modern global trend toward sustainable processes that meet the requirements of “green chemistry” provides new opportunities for the broad application of highly active, selective, and specific enzymatic reactions. However, the effective application of enzymes in industrial processes requires the development of systems for the remote regulation of their activity triggered by external physical stimuli, one of which is a low-frequency magnetic field (LFMF). Magnetic nanoparticles (MNPs) transform the energy of an LFMF into mechanical forces and deformations applied to enzyme molecules on the surfaces of MNPs. Here, we demonstrate the up- and down-regulation of two biotechnologically important enzymes, yeast alcohol dehydrogenase (YADH) and soybean formate dehydrogenase (FDH), in aggregates with gold-covered magnetic nanoparticles (GCMNPs) triggered by an LFMF. Two types of aggregates, “dimeric” (with the enzyme attached to several GCMNPs simultaneously), with YADH or FDH, and “monomeric” (the enzyme attached to only one GCMNP), with FDH, were synthesized. Depending on the aggregate type (“dimeric” or “monomeric”), LFMF treatment led to a decrease (down-regulation) or an increase (up-regulation) in enzyme activity. For “dimeric” aggregates, we observed 67 ± 9% and 47 ± 7% decreases in enzyme activity under LFMF exposure for YADH and FDH, respectively. Moreover, in the case of YADH, varying the enzyme or the cross-linking agent concentration led to different magnitudes of the LFMF effect, which was more significant at lower enzyme and higher cross-linking agent concentrations. Different responses to LFMF exposure depending on cofactor presence were also demonstrated. This effect might result from a varying cofactor binding efficiency to enzymes. For the “monomeric” aggregates with FDH, the LFMF treatment caused a significant increase in enzyme activity; the magnitude of this effect depended on the cofactor type: we observed up to 40% enzyme up-regulation in the case of NADP+, while almost no effect was observed in the case of NAD+.

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Section: Methodsmentioning

confidence: 99%

Up- and Down-Regulation of Enzyme Activity in Aggregates with Gold-Covered Magnetic Nanoparticles Triggered by Low-Frequency Magnetic Field

Veselov,

Efremova,

Prusov

et al. 2024

Nanomaterials

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“…In some cases, combination of mutations results in synergy. For example, such effect has been observed while improving thermal stability of soybean FDH [ 16 ]. In case of PseFDH SM4S mutant, replacements in 311th position generate enzymes with a 2.4-fold improved thermal stability with respect to the initial mutant, and more than 7-fold if compared to the wild-type PseFDH [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Effect of Additional Amino Acid Replacements on the Properties of Multi-point Mutant Bacterial Formate Dehyderogenase PseFDH SM4S

et al. 2022

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Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.

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Role of a Structurally Equivalent Phenylalanine Residue in Catalysis and Thermal Stability of Formate Dehydrogenases from Different Sources

Тишков

Goncharenko

Алексеева

et al. 2015

Biochemistry Moscow

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Comparison of amino acid sequences of NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2) from different sources and analysis of structures of holo-forms of FDH from bacterium Pseudomonas sp. 101 (PseFDH) and soya Glycine max (SoyFDH) as well as of structure of apo-form of FDH from yeast Candida boidinii (CboFDH) revealed the presence on the surface of protein globule of hydrophobic Phe residue in structurally equivalent position (SEP). The residue is placed in the coenzyme-binding domain and protects bound NAD+ from solvent. The effects of amino acid changes of the SEP on catalytic properties and thermal stability of PseFDH, SoyFDH, and CboFDH were compared. The strongest effect was observed for SoyFDH. All eight amino acid replacements resulted in increase in thermal stability, and in seven cases, increase in catalytic constant was achieved. Thermal stability of SoyFDH after mutations Phe290Asp and Phe290Glu increased 66- and 55-fold, respectively. KM values of the enzyme for substrate and coenzyme in different cases slightly increased or decreased. In case of one CboFDH, the mutein catalytic constant increased and thermal stability did not changed. In case of the second CboFDH mutant, results were the opposite. In one PseFDH mutant, amino acid change did not influence the catalytic constant, but in three others, the parameter was reduced. Two PseFDH mutants had higher and two mutants lower thermal stability in comparison with initial enzyme. Analysis of results of SEP mutagenesis in FDHs from bacterium, yeast, and plant shows that protein structure plays a key role for effect of the same amino acid changes in equivalent position in protein globule of formate dehydrogenases from different sources.

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Additivity of the Stabilization Effect of Single Amino Acid Substitutions in Triple Mutants of Recombinant Formate Dehydrogenase from the Soybean Glycine max

Cited by 5 publications

References 12 publications

Up- and Down-Regulation of Enzyme Activity in Aggregates with Gold-Covered Magnetic Nanoparticles Triggered by Low-Frequency Magnetic Field

Up- and Down-Regulation of Enzyme Activity in Aggregates with Gold-Covered Magnetic Nanoparticles Triggered by Low-Frequency Magnetic Field

Effect of Additional Amino Acid Replacements on the Properties of Multi-point Mutant Bacterial Formate Dehyderogenase PseFDH SM4S

Role of a Structurally Equivalent Phenylalanine Residue in Catalysis and Thermal Stability of Formate Dehydrogenases from Different Sources

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