Substrate promiscuity of cytochrome P450 RhF

O’Reilly, Elaine; Corbett, Mark; Hussain, Shahed; Kelly, Paul P.; Richardson, Donna; Flitsch, Sabine L.; Turner, Nicholas J.

doi:10.1039/c3cy00091e

Cited by 43 publications

(33 citation statements)

References 27 publications

(22 reference statements)

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“…4b,4d As a catalyst, we used the P411 BM3 -CIS T438S variant of cytochrome P450 BM3 , possessing the aforementioned C400S mutation. This enzyme, which contains 14 mutations relative to wild-type P450 BM3 (Table S1), was previously shown to be a good catalyst in the activation of azides for intramolecular C–H insertion.…”

Section: Resultsmentioning

confidence: 99%

Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer

et al. 2014

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Engineering enzymes with novel reaction modes promises to expand the applications of biocatalysis in chemical synthesis and will enhance our understanding of how enzymes acquire new functions. The insertion of nitrogen-containing functional groups into unactivated C–H bonds is not catalyzed by known enzymes but was recently demonstrated using engineered variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium. Here, we extend this novel P450-catalyzed reaction to include intermolecular insertion of nitrogen into thioethers to form sulfimides. An examination of the reactivity of different P450BM3 variants towards a range of substrates demonstrates that electronic properties of the substrates are important in this novel enzyme-catalyzed reaction. Moreover, amino acid substitutions have a large effect on the rate and stereoselectivity of sulfimidation, demonstrating that the protein plays a key role in determining reactivity and selectivity. These results provide a stepping-stone for engineering more complex nitrogen-atom transfer reactions in P450 enzymes and developing a more comprehensive biocatalytic repertoire.

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Section: Resultsmentioning

confidence: 99%

Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer

et al. 2014

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“…Usually, these reactions were restricted to perform in low substrate concentration due to the toxic and inhibitory caused by sulfide and sulfoxide. For instance, the P450SMO, P450RhF and P450BM3 variants were carried out with 1–2 mM sulfides. Though P450cam and CYP154H1 were reported to act on 4–5 mM sulfides, both gave relatively low conversion and stereoselectivity unfortunately.…”

Section: Figurementioning

confidence: 99%

Biocatalytical Asymmetric Sulfoxidation by Identifying Cytochrome P450 from Parvibaculum Lavamentivorans DS‐1

Tang

Cui

et al. 2018

ChemCatChem

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Cytochrome P450 monooxygenases (P450s) catalyzed asymmetric sulfoxidation represents a green route for the synthesis of valuable enantiopure sulfoxides, which are potentially interesting synthons in synthetic and pharmaceutical chemistry. Here the potential P450 and redox partner genes from Parvibaculum lavamentivorans DS‐1 are screened and co‐expressed in Escherichia coli host to construct twenty recombinant P450 strains. By testing the whole‐cell biooxidation of thioanisole, P450PL2 (CYP278A4) and P450PL7 (CYP108G3) are identified with excellent S enantioselectivity while P450PL1 (CYP111B1) and P450PL9 (CYP153A26) exhibit the complementary R enantioselectivity. Asymmetric sulfoxidation of sulfides 1 a–1 m is further investigated using the recombinant E. coli strain P450PL2‐2 based on the optimal conditions, producing the corresponding enantioenriched sulfoxides with up to 82 % isolated yield and 99 % ee.

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“…If an enzyme uses a great many substrates it is not very likely that it will be very discriminative of e.g., stereoisomers of the same molecule [although some transporters are stereoselective (Zhou et al, 2014), e.g., those for propranolol (Wang et al, 2010a; Zheng et al, 2013)]. By contrast, the activity of many promiscuous enzymes (e.g., the cytochromes P450, e.g., O'reilly et al, 2011, 2013; Munro et al, 2013) can be related (up to a cut-off) to the lipophilicity of the substrate. This is very simply explained in terms of the possession of a hydrophobic substrate pocket.…”

Section: Intellectual Challenges Around Bilayer Lipoidal Permeabilitymentioning

confidence: 99%

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

Kell¹,

Oliver²

2014

Front. Pharmacol.

147

169

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One approach to experimental science involves creating hypotheses, then testing them by varying one or more independent variables, and assessing the effects of this variation on the processes of interest. We use this strategy to compare the intellectual status and available evidence for two models or views of mechanisms of transmembrane drug transport into intact biological cells. One (BDII) asserts that lipoidal phospholipid Bilayer Diffusion Is Important, while a second (PBIN) proposes that in normal intact cells Phospholipid Bilayer diffusion Is Negligible (i.e., may be neglected quantitatively), because evolution selected against it, and with transmembrane drug transport being effected by genetically encoded proteinaceous carriers or pores, whose “natural” biological roles, and substrates are based in intermediary metabolism. Despite a recent review elsewhere, we can find no evidence able to support BDII as we can find no experiments in intact cells in which phospholipid bilayer diffusion was either varied independently or measured directly (although there are many papers where it was inferred by seeing a covariation of other dependent variables). By contrast, we find an abundance of evidence showing cases in which changes in the activities of named and genetically identified transporters led to measurable changes in the rate or extent of drug uptake. PBIN also has considerable predictive power, and accounts readily for the large differences in drug uptake between tissues, cells and species, in accounting for the metabolite-likeness of marketed drugs, in pharmacogenomics, and in providing a straightforward explanation for the late-stage appearance of toxicity and of lack of efficacy during drug discovery programmes despite macroscopically adequate pharmacokinetics. Consequently, the view that Phospholipid Bilayer diffusion Is Negligible (PBIN) provides a starting hypothesis for assessing cellular drug uptake that is much better supported by the available evidence, and is both more productive and more predictive.

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Substrate promiscuity of cytochrome P450 RhF

Cited by 43 publications

References 27 publications

Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer

Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer

Biocatalytical Asymmetric Sulfoxidation by Identifying Cytochrome P450 from Parvibaculum Lavamentivorans DS‐1

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

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