Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency

Darimont, Dominique; Weissenborn, Martin J.; Nebel, Bernd A.; Hauer, Bernhard

doi:10.1016/j.bioelechem.2017.08.009

Cited by 19 publications

(21 citation statements)

References 34 publications

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“…Using self‐sufficient P450s as a scaffold to construct chimeric enzymes has been proven a promising measure to produce functional P450 systems. In addition, modulating the electron transfer path to elevate the enzyme efficacy also showed promising effects in enhancing P450 performance [91] . Although a lot of effort has been made towards P450 domain engineering, modifying or fine‐tuning the entire electron traveling path is now possible owing to more structural information of length self‐sufficient P450s being available.…”

Section: Discussionmentioning

confidence: 99%

Advanced Understanding of the Electron Transfer Pathway of Cytochrome P450s

et al. 2020

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Cytochrome P450s are heme‐thiolate enzymes that participate in carbon source assimilation, natural compound biosynthesis and xenobiotic metabolism in all kingdoms of life. P450s can catalyze various reactions by using a wide range of organic compounds, thus exhibiting great potential in biotechnological applications. The catalytic reactions of P450s are driven by electron equivalents that are sourced from pyridine nucleotides and delivered by cognate or matching redox partners (RPs). The electron transfer (ET) route from RPs to P450s involves one or more redox center‐containing domains. As the rate of ET is one of the main determinants of P450 efficacy, an in‐depth understanding of the P450 ET pathway should increase our knowledge of these important enzymes and benefit their further applications. Here, the various P450 RP systems along with current understanding of their ET routes will be reviewed. Notably, state‐of‐the‐art structural studies of the two main types of self‐sufficient P450 will also be summarized.

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Section: Discussionmentioning

confidence: 99%

Advanced Understanding of the Electron Transfer Pathway of Cytochrome P450s

et al. 2020

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“…Comparing the identified residues with our HDX-MS approach shows that they align to the highly dynamic areas in the heme domain, as observed by comparing the heme domain to the full-length P450 BM3, shaded red and cyan in Figure 3 (36). Darimont et al mutated these residues (found within the K-and L-helices, as well as random coil segments of the heme domain and M490 of the FMN-binding domain) and found that electron transfer coupling efficiency and enzyme activity could be greatly altered by engineering these residues, but only when the redox reaction was driven by NADPH (the physiological cofactor), and not when an electrode system was used (37). Our data reveal areas that are not explained by these collective movements, but which exhibit some of the greatest changes in deuterium uptake.…”

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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“…For example, Dominique et al engineered the electrons transfer pathways of P450 BM3, resulting in a 32-fold improvement in coupling efficiency. [4] Hoffmann et al studied the linker region between CYP153 and reductase PFOR. [8] By increasing the linker by two amino acids, they obtained L3 variant with improved coupling efficiency (from 39 % to 94 %).…”

Section: Introductionmentioning

confidence: 99%

“…often leads to the inefficient use of expensive NAD(P)H cofactors and cause the accumulation of reactive oxygen species, represented by superoxide anion and hydrogen peroxide (H 2 O 2 ), which is toxic oxidant that damages the organisms. [4,7] Most studies have focused on improving catalytic performance. Generally, the increased coupling efficiency is accompanied by increased catalytic performance.…”

Section: Introductionmentioning

confidence: 99%

Modulating the Coupling Efficiency of P450 BM3 by Controlling Water Diffusion through Access Tunnel Engineering

Meng

Liu

et al. 2022

ChemSusChem

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Cytochromes P450 have gained much interest for their broad substrate scope in the catalysis of oxidation reactions for pharmaceuticals, plastics, and hormones. However, achieving high coupling efficiency by the engineering of P450s is still a big challenge. The presence of extra water around the active site is deemed to be related to uncoupling. In this study, the access tunnels of P450 BM3 from Bacillus megaterium are engineered to control water access from bulk solvent to the active site. Nine residues located in tunnels are investigated by site‐saturation mutagenesis to reduce water diffusion, thereby improving the coupling efficiency. The recombined variant N319L/T411V/T436A shows improved coupling efficiency (from 31.2 % to 52.6 %). Tunnel polarity analysis and molecular dynamics simulation further indicate that reduced water molecules around the active site lead to higher coupling efficiency. Overall, this study provides valuable insight on improving coupling efficiency by controlling water diffusion through tunnel engineering.

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Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency

Cited by 19 publications

References 34 publications

Advanced Understanding of the Electron Transfer Pathway of Cytochrome P450s

Advanced Understanding of the Electron Transfer Pathway of Cytochrome P450s

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

Modulating the Coupling Efficiency of P450 BM3 by Controlling Water Diffusion through Access Tunnel Engineering

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