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

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.

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

“…By using SaSrtA, the heme and reductase domain are reconstituted into the active and full-length P450 BM3 monooxygenase, which forms ad imer preventing ab ack reaction( Figure 1a). [34,35] The amount of reconstituted P450 BM3 was detectedb yareported fluorogenica ssay using 7benzoxy-3-carboxycoumarin ethyl ester (BCCE) as the substrate (Figure 1a). [36] Reconstitution of heme and reductase domain to full-length P450 BM3 using SaSrtA WT was previously reported.…”

Cascade reactions with two non‐physiological partners for NAD(P)H regeneration via renewable hydrogen

“…A dimerization was observed on the whole P450 BM3 of which BMR is a domain [ 70–73 ] but a direct BMR to BMR interaction was also observed under several conditions. [ 74,75 ] Here, a dimerization could compete with or hinder the productive interaction with CbA5H, leading to the strong decrease observed (Figure 3B).…”

Catalytically self‐sufficient CYP116B5: Domain switch for improved peroxygenase activity

“…The evidence suggests that in CYP116B5 the electron transfer chain may be less efficient than the one of other CYP116B subfamily members but it cannot be excluded that a different molecular mechanism takes place and that for this cytochrome the electron transfer relies more on the structural arrangement of the domains, which is supposed to be more dynamic and may involve less or no amino acid residues. [ 8,48 ] The fact that the reductase domain of CYP116B5 has the lowest cofactor conversion rate among the CYP116B isoenzymes, added to the evidence that the electron transfer chain may be less efficient than the one of other CYP116B isoenzymes. This is also in support of the assumption that CYP116B5 may have a different evolutionary path and that it is more related to peroxygenases rather than monooxygenases.…”