Rational Design of Practically Important Enzymes

Тишков, В. И.; Pometun, A. A.; Stepashkina, A. V.; Fedorchuk, V. V.; Zarubina, S. A.; Kargov, I.S.; Atroshenko, D. L.; Parshin, P. D.; Shelomov, M. D.; Kovalevski, R. P.; Boiko, K. M.; Eldarov, M. A.; D'Oronzo, Erica; Facheris, S.; Secundo, Francesco; Савин, С. С.

doi:10.3103/s0027131418020153

Cited by 27 publications

(16 citation statements)

References 22 publications

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“…According to Ju et al [23], mutant TvDAAOs with substitutions Met104Leu, Met156Leu and Met209Leu showed an increased stability towards hydrogen peroxide. However, our results for TvDAAO with the same substitutions were different [24,25]. It was found that substitutions Met104Leu, Met156Leu and Met209Leu did not change TvDAAO stability towards inactivation with hydrogen peroxide.…”

Section: Introductionmentioning

confidence: 77%

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

Atroshenko

Shelomov

Zarubina

et al. 2019

IJMS

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d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

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Section: Introductionmentioning

confidence: 77%

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

Atroshenko

Shelomov

Zarubina

et al. 2019

IJMS

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“…Molecular simulation methods, including quantum mechanics (QM), molecular mechanics (MM), and multiscale QM/MM modeling, have been extensively applied toward directed evolution to guide the selection of function-enhancing mutations . These simulations inform the mechanistic detail underlying the variation of rate and selectivity upon mutagenesis in an enzymatic reaction, inspiring the development of new design principles that pinpoint the beneficial mutations. − …”

Section: Introductionmentioning

confidence: 99%

EnzyHTP: A High-Throughput Computational Platform for Enzyme Modeling

Shao

Jiang

Yang

2022

J. Chem. Inf. Model.

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Molecular simulations, including quantum mechanics (QM), molecular mechanics (MM), and multiscale QM/MM modeling, have been extensively applied to understand the mechanism of enzyme catalysis and to design new enzymes. However, molecular simulations typically require specialized, manual operation ranging from model construction to data analysis to complete the entire life cycle of enzyme modeling. The dependence on manual operation makes it challenging to simulate enzymes and enzyme variants in a high-throughput fashion. In this work, we developed a Python software, EnzyHTP, to automate molecular model construction, QM, MM, and QM/MM computation, and analyses of modeling data for enzyme simulations. To test the EnzyHTP, we used fluoroacetate dehalogenase (FAcD) as a model system and simulated the enzyme interior electrostatics for 100 FAcD mutants with a random single amino acid substitution. For each enzyme mutant, the workflow involves structural model construction, 1 ns molecular dynamics (MD) simulations, and quantum mechanical calculations in 100 MD-sampled snapshots. The entire simulation workflow for 100 mutants was completed in 7 h with 10 GPUs and 160 CPUs. EnzyHTP improves the efficiency of computational enzyme modeling, setting a basis for high-throughput identification of function-enhancing enzymes and enzyme variants. The software is expected to facilitate the fundamental understanding of catalytic origins across enzyme families and to accelerate the optimization of biocatalysts for non-native substrates.

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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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Rational Design of Practically Important Enzymes

Cited by 27 publications

References 22 publications

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

EnzyHTP: A High-Throughput Computational Platform for Enzyme Modeling

Efficient Assay and Marker Significance of NAD+ in Human Blood

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