Comparison of Thermal Stability of New Formate Dehydrogenases by Differential Scanning Calorimetry

Pometun, A. A.; Kleymenov, Sergey Y.; Zarubina, S. A.; Kargov, I.S.; Parshin, P. D.; Садыхов, Э. Г.; Савин, С. С.; Тишков, В. И.

doi:10.3103/s002713141802013x

Cited by 11 publications

(3 citation statements)

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“…In the present study, we use FDH from Pseudomonas sp. that is extremely specific to NAD + , i.e., does not catalyze the reduction of NADP + , and is characterized by high catalytic efficiency and high thermal stability, compared to FDH from other sources ( 33 , 46 , 47 ). Employing this enzyme and developing the optimized protocol of the extraction of NAD + from blood, we demonstrate the diagnostic potential of the assay for medical application by measuring the whole blood NAD + in the healthy subjects and patients with neurological and cardiological pathologies.…”

Section: Introductionmentioning

confidence: 99%

Efficient Assay and Marker Significance of NAD+ in Human Blood

et al. 2022

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Oxidized nicotinamide adenine dinucleotide (NAD+) is a biological molecule of systemic importance. Essential role of NAD+ in cellular metabolism relies on the substrate action in various redox reactions and cellular signaling. This work introduces an efficient enzymatic assay of NAD+ content in human blood using recombinant formate dehydrogenase (FDH, EC 1.2.1.2), and demonstrates its diagnostic potential, comparing NAD+ content in the whole blood of control subjects and patients with cardiac or neurological pathologies. In the control group (n = 22, 25–70 years old), our quantification of the blood concentration of NAD+ (18 μM, minimum 15, max 23) corresponds well to NAD+ quantifications reported in literature. In patients with demyelinating neurological diseases (n = 10, 18–55 years old), the NAD+ levels significantly (p < 0.0001) decrease (to 14 μM, min 13, max 16), compared to the control group. In cardiac patients with the heart failure of stage II and III according to the New York Heart Association (NYHA) functional classification (n = 24, 42–83 years old), the blood levels of NAD+ (13 μM, min 9, max 18) are lower than those in the control subjects (p < 0.0001) or neurological patients (p = 0.1). A better discrimination of the cardiac and neurological patients is achieved when the ratios of NAD+ to the blood creatinine levels, mean corpuscular volume or potassium ions are compared. The proposed NAD+ assay provides an easy and robust tool for clinical analyses of an important metabolic indicator in the human blood.

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Section: Introductionmentioning

confidence: 99%

Efficient Assay and Marker Significance of NAD+ in Human Blood

et al. 2022

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“…Crystal structures for apo- and holo-forms of PseFDH have been determined (PDB2NAC, PDB2NAD, PDB2GO1, and PDB2GUG structures). Despite the fact that many novel formate dehydrogenases have been cloned, isolated, and characterized in the last decades, PseFDH is still the one with the highest thermal stability [ 4 ], and high catalytic activity and efficiency [ 5 , 6 ]. Formate dehydrogenase from pathogenic bacterium Staphylococcus aureus (SauFDH) has been recently isolated and crystallized in this laboratory [ 7 ]; this enzyme is comparable to PseFDH in its thermal stability [ 4 ] and exhibits a higher catalytic activity, but not efficiency [ 6 ].…”

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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“…PseFDH -первая бактериальная формиатдегидрогеназа, ген которой был клонирован и успешно экспрессирован в клетках E. coli [2,3]. Несмотря на то, что за последние пятьдесят с лишним лет было описано много новых ФДГ из различных источников, PseFDH до сих остается ферментом с самой высокой термостабильностью среди ферментов этой группы [4]. Кроме того, как и большинство бактериальных формиатдегидрогеназ, PseFDH имеет более высокую каталитическую константу по сравнению с ФДГ из эукариот [5,6].…”

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Highly Stable Mutant Bacterial Format Dehydrogenase With Improved Catalytic Properties

Pometun

Shirokova

G.P.

et al. 2023

Lomonosov Chemistry Journal

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NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from methylotrophic bacterium Pseudomonas sp.101 (PseFDH) has one of the highest thermal stability among all known enzymes of this group. The introduction of a number of amino acid substitutions into PseFDH made it possible to obtain a multipoint mutant PseFDH SM4S enzyme with even higher temperature and chemical stability. Previously, we showed that the introduction of additional single point replacements S131A, or S160A, or E170D into PseFDH SM4S led to further stabilization of the enzyme. In this work, based on the PseFDH SM4S S131A mutant, new mutant FDHs obtained, in which, compared to PseFDH SM4S, we added double S131A/E170D (M2), triple S131A/S160A/E170D (M3) and quadruple S131A/S160A/ E170D/S145A (PseFDH SM4A M3) amino acids replacements. The new PseFDH mutants were overexpressed in E. coli cells, puri ed and characterized. The S131A/E170D and S131A/S160A/E170D changes provided further improving thermal stability. The introduction of the S145A substitution into PseFDH SM4S M4 leads to a signi cant decrease in KMNAD+ and KMHCOO- while maintaining the catalytic constant at the same level. This mutant form can be successfully used in NADH regeneration systems, as well as for the detection of NAD+ and formate in biological systems.

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Comparison of Thermal Stability of New Formate Dehydrogenases by Differential Scanning Calorimetry

Cited by 11 publications

References 11 publications

Efficient Assay and Marker Significance of NAD+ in Human Blood

Efficient Assay and Marker Significance of NAD+ in Human Blood

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

Highly Stable Mutant Bacterial Format Dehydrogenase With Improved Catalytic Properties

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