Full Structure Modeling of Three-Domains Monooxygenase CYP102A1 ВМ3 from Bacillus megaterium

Krivitskaya, Alexandra V.; Pometun, A. A.; Parshin, P. D.; Khrenova, Maria G.; Urlacher, Vlada B.; Тишков, В. И.

doi:10.3103/s0027131420030074

Cited by 3 publications

(1 citation statement)

References 19 publications

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“…This phenomenon, termed uncoupling, is the cause of insufficient electron transfer between enzyme domains or unfavorable substrate binding [11] . As member of the class VIII P450s, the P450 BM3 subunits’ modular structure consists of two domains: [12,13] the C‐terminal NAD(P)H‐dependent diflavin‐reductase domain (471–1048) containing flavin mononucleotide (FMN) as well as flavin adenine dinucleotide (FAD) for electron transfer, and the N‐terminal catalytic heme domain (1–470) for hydroxylation of the substrate. Both domains are fused as a single polypeptide making the P450 BM3 independent of external redox partners which is referred to as self‐sufficiency [7,14] .…”

Section: Introductionmentioning

confidence: 99%

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification

Essert,

Castiglione

2023

ChemBioChem

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The vast majority of known enzymes exist as oligomers, which often gives them high catalytic performance but at the same time imposes constraints on structural conformations and environmental conditions. An example of an enzyme with a complex architecture is the P450 BM3 monooxygenase CYP102A1 from Bacillus megaterium. Only active as a dimer, it is highly sensitive to dilution or common immobilization techniques. In this study, we engineered a thermostable P450BM3 chimera consisting of the heme domain of a CYP102A1 variant and the reductase domain of the homologous CYP102A3. The dimerization of the hybrid was even weaker compared to the corresponding CYP102A1 variant. To create a stable dimer, we covalently coupled the C‐termini of two monomers of the chimera via SpyTag003/SpyCatcher003 interaction. As a result, purification, thermostability, pH stability, and catalytic activity were improved. Via a bioorthogonal two‐step affinity purification, we obtained high purity (94 %) of the dimer‐stabilized variant being robust against heme depletion. Long‐term stability was increased with a half‐life of over 2 months at 20 °C and 80–90 % residual activity after 2 months at 5 °C. Most catalytic features were retained with even an enhancement of the overall activity by ~2‐fold compared to the P450BM3 chimera without SpyTag003/SpyCatcher003.

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Section: Introductionmentioning

confidence: 99%

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification

Essert,

Castiglione

2023

ChemBioChem

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Formate Dehydrogenase: From NAD(P)H Regeneration to Targeting Pathogen Biofilms, Composing Highly Efficient Hybrid Biocatalysts and Atmospheric CO2 Fixation

Tishkov,

Pometun,

Savin

2023

Moscow Univ. Chem. Bull.

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Formate Dehydrogenase: From Nad(p)h Regeneration To the Target in Pathogens Biofilms, a Component Of Highly Efficient Hybrid Biocatalysts and Co2 Fixation From the Atmosphere

Tishkov,

Pometun,

Savin

2023

Lomonosov Chemistry Journal

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NAD(P)+ -dependent formate dehydrogenase (EC 1.2.1.2, FDH) catalyzes the simple from chemical and biological point of view reaction of formate ion oxidation to carbon dioxide with corresponding reduction of NAD(P)+ to NAD(P) H. Advances in the life sciences have shown that this reaction plays an extremely important role in a wide variety of organisms. The areas and types of practical applications of FDH are also permanently expanding. In this review we considered the main stages in the development of understanding and knowledge about the role of formate dehydrogenase in living systems. Achievements in creation of highly effi cient catalysts based on FDH for classic biotechnology as well as for new areas are also considered. The importance of appropriate choice of the initial FDH for the creation of a biocatalyst with the required and prescribed properties with minimal costs is shown. The prospects for the use of FDH for the fixation of CO2 are discussed.

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Full Structure Modeling of Three-Domains Monooxygenase CYP102A1 ВМ3 from Bacillus megaterium

Cited by 3 publications

References 19 publications

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification

Formate Dehydrogenase: From NAD(P)H Regeneration to Targeting Pathogen Biofilms, Composing Highly Efficient Hybrid Biocatalysts and Atmospheric CO2 Fixation

Formate Dehydrogenase: From Nad(p)h Regeneration To the Target in Pathogens Biofilms, a Component Of Highly Efficient Hybrid Biocatalysts and Co2 Fixation From the Atmosphere

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