Redox Partner Interaction Sites in Cytochrome P450 Monooxygenases:<i>In Silico</i>Analysis and Experimental Validation

Gricman, Łukasz; Weissenborn, Martin J.; Hoffmann, Sara M.; Borlinghaus, Niels; Hauer, Bernhard; Pleiss, Jürgen

doi:10.1002/slct.201600369

Cited by 10 publications

(6 citation statements)

References 65 publications

(130 reference statements)

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“…has been fused to the cytochrome P450 reductase (CPR) domain of CYP102A1 (BM3), the PFOR domain of CYP116B3 [13] and CYP116B2 [10]. This is however no trivial task, as not only do the linker region's sequence and length significantly influence the activity and coupling efficiency [10] but given that these reductase partners are not the CYPs' natural redox partners, interdomain contacts also have to be optimized [14]. Although higher coupling efficiencies have been observed with artificial fusion systems such as CYP153A35 fused to the CPR of P450BM3, CYP153A35 displayed higher productivities in whole cells when used as a three-component system in combination with putidaredoxin (CamB) and putidaredoxin reductase (CamA) [15].…”

Section: Introductionmentioning

confidence: 99%

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Kochius¹,

Marwijk²,

Ebrecht³

et al. 2018

Catalysts

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Some of the most promising results for bacterial alkane hydroxylation to alcohols have been obtained with the cytochrome P450 monooxygenase CYP153A6. CYP153A6 belongs to the class I CYPs and is generally expressed from an operon that also encodes the ferredoxin (Fdx) and ferredoxin reductase (FdR) which transfer electrons to CYP153A6. In this study, purified enzymes (CYP, Fdx, FdR and dehydrogenases for cofactor regeneration) were used to deconstruct the CYP153A6 system into its separate components, to investigate the factors limiting octane hydroxylation in vitro. Proteins in the cytoplasm (cell-free extract) were found to better enhance and stabilize hydroxylase activity compared to bovine serum albumin (BSA) and catalase. Optimization of the CYP:Fdx:FdR ratio also significantly improved both turnover frequencies (TFs) and total turnover numbers (TTNs) with the ratio of 1:1:60 giving the highest values of 3872 h−1 and 45,828 moloctanol molCYP−1, respectively. Choice and concentration of dehydrogenase for cofactor regeneration also significantly influenced the reaction. Glucose dehydrogenase concentrations had to be as low as possible to avoid fast acidification of the reaction medium, which in the extreme caused precipitation of the CYP and other proteins. Cofactor regeneration based on glycerol failed, likely due to accumulation of dihydroxyacetone. Scaling the reactions up from 1 mL in vials to 60 mL in shake flasks and 120 mL in bioreactors showed that mixing and shear forces will be important obstacles to overcome in preparative scale reactions.

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Section: Introductionmentioning

confidence: 99%

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Kochius¹,

Marwijk²,

Ebrecht³

et al. 2018

Catalysts

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“…7 The proximal face of a P450 is also the site where interactions with redox partner proteins occur, with these processes largely appearing to be driven by electrostatic interactions. 8 The active site of P450s comprises regions that surround the distal face of the heme moiety, and include the so-called BC loop region between the B-and C-helices, the C-terminal region of the F-helix, the N-terminal portion of the G-helix, the centre of the I-helix, the b-strand following the K-helix and the C-terminal loop that impinges of the P450 active site (such regions are also referred to as substrate recognition sites, SRSs 1-6). 9 Variability within these regions leads to the ability of P450s to accept a wide range of different substrates (even those that are protein bound), whilst the retention of the general structure surrounding the active site heme group and axial cysteine ligand support the remarkable consistency of the P450 catalytic mechanism.…”

Section: 3mentioning

confidence: 99%

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

et al. 2018

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The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations. In this review, we aim to provide an overview of the range of chemical transformations that P450 enzymes can catalyse within bacterial secondary metabolism, with the intention to provide an important resource to aid in understanding of the potential roles of P450 enzymes within newly identified bacterial biosynthetic pathways. 1.Introduction to P450s 2.Chemistry of P450 enzymes 3.Bacterial biosynthesis pathways involving P450s 3.1Terpene metabolism 3.1. Battersby (1925Battersby ( -2018 to the molecular understanding of biosynthesis over the course of their careers.

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“…Computational analysis indicated that electron transfer from FMN-toheme in the conformation observed in the crystal structure would take ~50 years to complete for an extended ~50 σ-bond route (through amino acids and their peptide bonds) from FMN-to-heme. This is unlikely to be an efficient electron tunnelling pathway, and an alternative route of ~18 Å between conjugated edges of the FMN cofactor and the heme prosthetic group was calculated to have an electron transfer rate constant of ~12 s -1 (1,16,35). Despite this, modelling of the interactions between the BM3 heme domain and the FMN domain identifies larger regions of interest from the 1BVY structure.…”

Section: Discussionmentioning

confidence: 99%

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

Jeffreys

Pacholarz

Johannissen

et al. 2020

Journal of Biological Chemistry

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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.

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Redox Partner Interaction Sites in Cytochrome P450 Monooxygenases:In SilicoAnalysis and Experimental Validation

Cited by 10 publications

References 65 publications

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

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

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