Engineering a self-sufficient Mycobacterium tuberculosis CYP130 by gene fusion with the reductase-domain of CYP102A1 from Bacillus megaterium
| Autor: | Nico P. E. Vermeulen, Daan P. Geerke, Sandra Ortega Ugalde, Jan N. M. Commandeur, Wilbert Bitter, Rosa A. Luirink |
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| Přispěvatelé: | Molecular and Computational Toxicology, AIMMS, Molecular Microbiology |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Recombinant Fusion Proteins Molecular Dynamics Simulation Reductase Dextromethorphan Methylation Biochemistry Catalysis Substrate Specificity Inorganic Chemistry Mycobacterium tuberculosis Inhibitory Concentration 50 03 medical and health sciences Bacterial Proteins Cytochrome P-450 Enzyme System SDG 3 - Good Health and Well-being Humans Cloning Molecular NADPH-Ferrihemoprotein Reductase Bacillus megaterium chemistry.chemical_classification 030102 biochemistry & molecular biology biology Active site Protein engineering biology.organism_classification Fusion protein Molecular Docking Simulation Kinetics 030104 developmental biology Enzyme chemistry Docking (molecular) biology.protein Gene Fusion Oxidation-Reduction |
| Zdroj: | Ortega Ugalde, S, Luirink, R A, Geerke, D P, Vermeulen, N P E, Bitter, W & Commandeur, J N M 2017, ' Engineering a self-sufficient Mycobacterium tuberculosis CYP130 by gene fusion with the reductase-domain of CYP102A1 from Bacillus megaterium ', Journal of Inorganic Biochemistry, vol. 180, 180, pp. 48 . https://doi.org/10.1016/j.jinorgbio.2017.12.003 Journal of Inorganic Biochemistry, 180:180. Elsevier Inc. |
| ISSN: | 0162-0134 |
| DOI: | 10.1016/j.jinorgbio.2017.12.003 |
| Popis: | CYP130 belongs to the subset of cytochrome P450s from Mycobacterium tuberculosis (Mtb) that have been structurally characterized. Despite several efforts for its functional characterization, CYP130 is still considered an orphan enzyme for which no endogenous or exogenous substrate has been identified. In addition, functional redox-partners for CYP130 have not been clearly established yet, hampering the elucidation of its physiological role. In the present study, a catalytically active fusion protein involving CYP130 and the NADPH reductase-domain of CYP102A1 from Bacillus megaterium was created. By screening a panel of known substrates of human P450s, dextromethorphan N-demethylation was identified as a reaction catalyzed by CYP130. The fusion enzyme showed higher catalytic activity, when compared to CYP130 reconstituted with a selection of non-native redox-partners. Molecular dynamics simulation studies based on the crystal structure of CYP130 revealed two primary docking poses of dextromethorphan within the active site consistent with the experimentally observed N-demethylation reaction during the entire molecular dynamics simulation. The dextromethorphan N-demethylation reaction was strongly inhibited by azole-drugs and maybe applied to identify mechanism-based inhibitors of CYP130. Furthermore, the present active CYP130-fusion protein may facilitate the identification of endogenous substrates from Mtb. |
| Databáze: | OpenAIRE |
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