Catalytic Properties and Stability of a Pseudomonas sp.101 Formate Dehydrogenase Mutants Containing Cys-255-Ser and Cys-255-Met Replacements

Тишков, В. И.; Galkin, Andrey; Марченко, Г. Н.; Egorova, O.; Sheluho, Dmitry; Kulakova, Ljudmila; Dementieva, L.A.; Егоров, А.М.

doi:10.1006/bbrc.1993.1511

Cited by 54 publications

(48 citation statements)

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“…101 and is significantly superior to the latter in chemical stability due to Val residue in position In 1993 in this laboratory (Faculty of Chemistry, Lomonosov Moscow State University) mutant FDHs were prepared from Pseudomonas sp. 101 with substitu tions Cys255Ser and Cys255Met [99]. These mutants dis played at least two order of magnitude higher chemical stability than the wild type enzyme.…”

Section: Application Of Formate Dehydrogenasementioning

confidence: 96%

Catalytic mechanism and application of formate dehydrogenase

Тишков

Popov

2004

Biochemistry (Moscow)

159

129

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NAD+-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energy supply of methylotrophic microorganisms and in response to stress in plants. FDH belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases. FDH is widely accepted as a model enzyme to study the mechanism of hydride ion transfer in the active center of dehydrogenases because the reaction catalyzed by the enzyme is devoid of proton transfer steps and implies a substrate with relatively simple structure. FDH is also widely used in enzymatic syntheses of optically active compounds as a versatile biocatalyst for NAD(P)H regeneration consumed in the main reaction. This review covers the late developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiological role, and its application for new chiral syntheses.

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Section: Application Of Formate Dehydrogenasementioning

confidence: 96%

Catalytic mechanism and application of formate dehydrogenase

Тишков

Popov

2004

Biochemistry (Moscow)

159

129

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“…Further details on reductase mutants will be described elsewhere. [52] The CYP102A1 reductase double mutant R966D, W1046S was fused to the CYP102A1 A74G, F87V, L188Q monooxygenase domain.To proof applicability of this NADH-dependent CYP102A1 mutant in organic synthesis, oxidation of myristic acid was performed in the two-phase reaction system described above for the NADPH-dependent variant, except that NADP + -dependent FDH and NADP + were exchanged by NAD + -dependent FDH [46] and NAD + . Extraction of the reaction mixture and subsequent GC/MS analysis revealed 20 % conversion to hydroxylated products.…”

Section: Conversion Of Myristic Acid By An Nadh-dependent Cyp102a1 Mumentioning

confidence: 99%

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP + -dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD + -dependent FDH was used.Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.

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“…The temperature stability of the muteins was slightly reduced compared to the wild type [7,12]. A similar observation was reported by Tishkov et al [11] for the bacterial FDH mutein.…”

Section: Formate Dehydrogenasesupporting

confidence: 76%

“…Using the crystal structure of Pseudomonas FDH as a model, both cysteines are likely to be accessible at the surface of the protein, but placed too far apart to form intramolecular disulfide bridges in the native molecule. Thishkov et al had reported that replacement of cysteine in the bacterial FDH by serine or methione had dramatically increased the resistance of Pseudomonas FDH against treatment with [11]. We decided therefore to replace Cys23 and Cys262 in C. boidinii FDH by small aliphatic amino acids using the appropriate genetic techniques and analyse for resistance of the mutants generated against oxidative stress.…”

Section: Formate Dehydrogenasementioning

confidence: 97%

Improvements of Enzyme Stability and Specificity by Genetic Engineering

Pohl

Kula

Engineering and Manufacturing for Biotechnology

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Catalytic Properties and Stability of a Pseudomonas sp.101 Formate Dehydrogenase Mutants Containing Cys-255-Ser and Cys-255-Met Replacements

Cited by 54 publications

References 0 publications

Catalytic mechanism and application of formate dehydrogenase

Catalytic mechanism and application of formate dehydrogenase

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Improvements of Enzyme Stability and Specificity by Genetic Engineering

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