Chimeric self-sufficient P450cam-RhFRed biocatalysts with broad substrate scope

Robin, Aélig; Köhler, Valentin; Jones, Alison S.; Ali, Afruja; Kelly, Paul P.; O’Reilly, Elaine; Turner, Nicholas J.; Flitsch, Sabine L.

doi:10.3762/bjoc.7.173

Cited by 39 publications

(36 citation statements)

References 27 publications

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“…By analogy to P450cam 47,48 and the human P450 3A4 49 , reorganization of the MycG active site may be driven by binding of the redox partner. As has been recently demonstrated by elegant work from the Poulos laboratory 35 , binding of the endogenous redox partner putidaredoxin stabilizes the “open” conformation of P450cam, facilitating substrate access to the active site.…”

Section: Discussionmentioning

confidence: 87%

“…Motivated by our recent work 14-16 and that of others 32-35 in enhancing catalytic activity by fusing the RhFRED reductase domain to the C-terminus of various biosynthetic P450s, we constructed the MycG-RhFRED fusion protein. Similar to MycG supported by separate spinach Fdx/FdR, this single-component self-sufficient P450 system driven by NADPH, achieved all physiological reactions known for MycG (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

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New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners

et al. 2014

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Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

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Section: Discussionmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners

et al. 2014

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“…Previous work demonstrated that Baeyer–Villiger monooxygenases could be genetically fused with NADP + reductases, which simplified cofactor regeneration . Ferrodoxin and flavodoxin reductase‐type domains could also be fused to cytochrome P450 heme domains to generate self‐sufficient hydroxylation catalysts . We therefore envisioned that an FDH‐FR fusion enzyme could be useful for a wide range of in vitro and in vivo applications.…”

Section: Methodsmentioning

confidence: 99%

Aromatic Halogenation by Using Bifunctional Flavin Reductase–Halogenase Fusion Enzymes

et al. 2017

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The remarkable site-selectivity and broad substrate scope of flavin dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site-selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase (FR), is required. Here, we report functional synthetic, fused FDH-FR proteins, containing various FDHs and FRs, joined by different linkers. We show that FDH-FR fusion proteins can increase product titers compared to the individual components for in vivo biocatalysis in E. coli.

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“…Increasing the length of the N-terminus may improve enzyme activity because excessive truncation can inhibit heme incorporation and correct heme pocket folding [64]. The versatile RhF reductase domain may also prove useful because this has been previously successfully combined with P450s from other sources [76,77]. X-ray diffraction data provide a more rational basis for the design of P450-CPR fusion proteins, but no crystal structures are yet available for the insect P450s [64].…”

Section: Discussionmentioning

confidence: 99%

Strategies for the construction of insect P450 fusion enzymes

Talmann

Wiesner

Vilcinskas

2017

Zeitschrift Für Naturforschung C

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Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with a broad substrate spectrum. Insect P450s are known to catalyze reactions such as the detoxification of insecticides and the synthesis of hydrocarbons, which makes them useful for many industrial processes. Unfortunately, it is difficult to utilize P450s effectively because they must be paired with cytochrome P450 reductases (CPRs) to facilitate electron transfer from reduced nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, eukaryotic P450s and CPRs are membrane-anchored proteins, which means they are insoluble and therefore difficult to purify when expressed in their native state. Both challenges can be addressed by creating fusion proteins that combine the P450 and CPR functions while eliminating membrane anchors, allowing the production and purification of soluble multifunctional polypeptides suitable for industrial applications. Here we discuss several strategies for the construction of fusion enzymes combining insect P450 with CPRs.

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Chimeric self-sufficient P450cam-RhFRed biocatalysts with broad substrate scope

Abstract: SummaryA high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of substrates was developed, leading to the identification of a number of novel biooxidation activities.

Cited by 39 publications

References 27 publications

New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners

New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners

Aromatic Halogenation by Using Bifunctional Flavin Reductase–Halogenase Fusion Enzymes

Strategies for the construction of insect P450 fusion enzymes

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