Influence of His6 Sequence on the Properties of Formate Dehydrogenase from Bacterium Pseudomonas sp. 101

Pometun, A. A.; Parshin, P. D.; Galanicheva, N. P.; Uporov, I. V.; Atroshenko, D. L.; Савин, С. С.; Тишков, В. И.

doi:10.3103/s0027131420040057

Cited by 16 publications

(6 citation statements)

References 13 publications

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“…One of the NADP +dependent FDHs is an enzyme from the bacteria Burkholderia stabilis (BstFDH), which has a positively charged Gln residue in this position 15 . It was shown in PseFDH that substitutions of Asp221Ser and Asp221Gln (221 amino acid residues are structurally equivalent to Asp218 in PseFDH) lead to the formation of an NADP + -dependent enzyme 16,17 . We decided to replace Asp218 to Gln in SauFDH to qualitatively assess whether this substitution would lead to a change in coenzyme specificity.…”

Section: Choice Of Mutationsmentioning

confidence: 99%

Improvement of the catalytic properties of formate dehydrogenase from bacterium Staphylococcus aureus by rational design.

A.A.,

T.S.,

S.B.

et al. 2023

Preprint

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NAD -dependent formate dehydrogenase from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of these bacteria. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. Since this enzyme has the highest k value among the described FDHs and also has a high temperature stability compared to other proteins, it can be considered as a promising catalyst for regeneration of NAD(P)H. The main disadvantage of this enzyme are high K values. In this work, the principle of rational design was used to reduce K . As a result, 9 catalytically significant positions 119, 194, 196, 217-219, 246, 303, and 323 were identified, and 16 new mutant forms of SauFDH were obtained and fully characterized. Substitutions in positions 119 and 194 lead to an increase of K . In the Ile-Val-Ala-Gly line, position 119 tends to improve NAD binding. K of SauFDH V119G decreased by 27 times compared to the wild-type enzyme. K Phe194Val decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. The use of a complex buffer increased the catalytic efficiency 6 times for the mutant with the Phe194Val substitution compared to wt-SauFDH in a single-component buffer.

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Section: Choice Of Mutationsmentioning

confidence: 99%

Improvement of the catalytic properties of formate dehydrogenase from bacterium Staphylococcus aureus by rational design.

A.A.,

T.S.,

S.B.

et al. 2023

Preprint

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show abstract

“…The experiments aimed at changing the coenzyme specificity have been initiated [ 11 ]; the mutants with changed isoelectric point have been constructed [ 12 ]. The effect of N-terminal His-tag on the properties of the wild-type enzyme and its NADP + -specific mutants has been studied by site-directed mutagenesis [ 13 ]. Chemical stability of PseFDH has been improved as well, and the mutants with an increased stability in the presence of hydrogen peroxide have been produced [ 14 , 15 ].…”

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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“…Получены мутантные формы с измененной изоэлектрической точкой [19]. Методом направленного мутагенеза исследовано влияние His-tag на N-конце аминокислотной последовательности на свойства PseFDH и NADP + -специфичных мутантов [20]. Для PseFDH проведены также эксперименты по увеличению химической стабильности.…”

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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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Influence of His6 Sequence on the Properties of Formate Dehydrogenase from Bacterium Pseudomonas sp. 101

Cited by 16 publications

References 13 publications

Improvement of the catalytic properties of formate dehydrogenase from bacterium Staphylococcus aureus by rational design.

Improvement of the catalytic properties of formate dehydrogenase from bacterium Staphylococcus aureus by rational design.

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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