Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.

Autor:	Jeffreys LN; The Manchester Institute of Biotechnology, School of Natural Sciences, Department of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.; Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom., Pacholarz KJ; Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom., Johannissen LO; The Manchester Institute of Biotechnology, School of Natural Sciences, Department of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.; Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom., Girvan HM; The Manchester Institute of Biotechnology, School of Natural Sciences, Department of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.; Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom., Barran PE; The Manchester Institute of Biotechnology, School of Natural Sciences, Department of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.; Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.; Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom., Voice MW; Cypex Ltd., 6 Tom McDonald Avenue, Dundee, DD2 1NH, United Kingdom., Munro AW; The Manchester Institute of Biotechnology, School of Natural Sciences, Department of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom andrew.munro@manchester.ac.uk.; Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2020 May 29; Vol. 295 (22), pp. 7595-7607. Date of Electronic Publication: 2020 Apr 17.
DOI:	10.1074/jbc.RA119.011630
Abstrakt:	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. (© 2020 Jeffreys et al.)
Databáze:	MEDLINE
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