Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches
| Autor: | Laura N. Jeffreys, Michael W. Voice, Hazel M. Girvan, Linus O. Johannissen, Andrew W. Munro, Kamila J. Pacholarz, Perdita E. Barran |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Dimer Crystallography X-Ray Biochemistry Mass Spectrometry chemistry.chemical_compound Cytochrome P-450 Enzyme System structural biology solution structure chemistry.chemical_classification dimerization native ion mobility mass spectrometry (IM-MS) biology Protein dynamics collision induced unfolding (CIU) Enzyme structure enzyme structure hydrogen-deuterium exchange mass spectrometry (HDX-MS) protein dynamics cytochrome P450 Stereochemistry protein solvent accessibility enzyme catalysis Cofactor Enzyme catalysis 03 medical and health sciences Bacterial Proteins Protein Domains flavoprotein Manchester Institute of Biotechnology fusion protein mass spectrometry (MS) monooxygenase Protein Structure Quaternary Molecular Biology NADPH-Ferrihemoprotein Reductase cytochrome P450 BM3 030102 biochemistry & molecular biology Deuterium Exchange Measurement Cell Biology ResearchInstitutes_Networks_Beacons/manchester_institute_of_biotechnology Fusion protein 030104 developmental biology Enzyme Structural biology chemistry reductase domain interactions Bacillus megaterium Enzymology biology.protein Protein Multimerization |
| Zdroj: | Jeffreys, L N, Pacholarz, K J, Johannissen, L O, Girvan, H M, Barran, P E, Voice, M W & Munro, A W 2020, ' Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches ', Journal of Biological Chemistry, vol. 295, no. 22, pp. 7595-7607 . https://doi.org/10.1074/jbc.RA119.011630 The Journal of Biological Chemistry |
| DOI: | 10.1074/jbc.RA119.011630 |
| Popis: | The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3. |
| Databáze: | OpenAIRE |
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