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

Laura N. Jeffreys, Kamila J. Pacholarz, Linus O. Johannissen, Hazel M. Girvan, Perdita E. Barran, Michael W. Voice, Andrew W. Munro

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)7595-7607
Number of pages13
JournalJournal of Biological Chemistry
Volume295
Issue number22
Early online date17 Apr 2020
DOIs
Publication statusPublished - 29 May 2020
Externally publishedYes

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