Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion.
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ABSTRACT:
SUBMITTER: Saroay R
PROVIDER: S-EPMC8359946 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB'1 helix of the protein to seal the binding pocket.
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Project description:Thaxtomins, a family of phytotoxins produced by Streptomyces spp., can cause dramatic plant cell hypertrophy and seedling stunting. Thaxtomin A is the dominant form from Streptomyces scabies and has demonstrated herbicidal action. TxtE, a cytochrome P450 enzyme from Streptomyces scabies 87.22, catalyzes direct nitration of the indolyl moiety of L-tryptophan to L-4-nitrotryptophan using nitric oxide, dioxygen and NADPH. The crystal structure of TxtE was determined at 2.1 Å resolution and described in this work. A clearly defined substrate access channel is observed and can be classified as channel 2a, which is common in bacteria cytochrome P450 enzymes. A continuous hydrogen bond chain from the active site to the external solvent is observed. Compared with other cytochrome P450 enzymes, TxtE shows a unique proton transfer pathway which crosses the helix I distortion. Polar contacts of Arg59, Tyr89, Asn293, Thr296, and Glu394 with L-tryptophan are seen using molecular docking analysis, which are potentially important for substrate recognition and binding. After mutating Arg59, Asn293, Thr296 or Glu394 to leucine, the substrate binding ability of TxtE was lost or decreased significantly. Based on the docking and mutation results, a possible mechanism for substrate recognition and binding is proposed.
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Project description:The cytochrome P450 reductase (POR) transfers electrons to all microsomal cytochrome P450 enzymes (CYP450) thereby driving their activity. In the vascular system, the POR/CYP450 system has been linked to the production of epoxyeicosatrienoic acids (EETs) but also to the generation of reactive oxygen species. In cardiac myocytes (CMs), EETs have been shown to modulate the cardiac function and have cardioprotective effects. The functional importance of the endothelial POR/CYP450 system in the heart is unclear and was studied here using endothelial cell-specific, inducible knockout mice of POR (ecPOR-/-). RNA sequencing of murine cardiac cells revealed a cell type-specific expression of different CYP450 homologues. Cardiac endothelial cells mainly expressed members of the CYP2 family which produces EETs, and of the CYP4 family that generates omega fatty acids. Tamoxifen-induced endothelial deletion of POR in mice led to cardiac remodelling under basal conditions, as shown by an increase in heart weight to body weight ratio and an increased CM area as compared to control animals. Endothelial deletion of POR was associated with a significant increase in endothelial genes linked to protein synthesis with no changes in genes of the oxidative stress response. CM of ecPOR-/- mice exhibited attenuated expression of genes linked to mitochondrial function and an increase in genes related to cardiac myocyte contractility. In a model of pressure overload (transverse aortic constriction, TAC with O-rings), ecPOR-/- mice exhibited an accelerated reduction in cardiac output (CO) and stroke volume (SV) as compared to control mice. These results suggest that loss of endothelial POR along with a reduction in EETs leads to an increase in vascular stiffness and loss in cardioprotection, resulting in cardiac remodelling.
2022-12-21 | GSE214776 | GEO
Project description:Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and Escherichia coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe substrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3's higher k(cat) seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR (K(D) and K(m,red)) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher k(cat). In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in k(cat) between the two enzymes, and may provide an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level.
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Project description:The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.
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Project description:Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.
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Project description:Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the "140s" FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the "140s loop" by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the "140s loop".
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Project description:Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein-protein docking to investigate the conformational changes necessary for the formation of the CPR-CYP complex. While some structural features of the CPR and the CPR-CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR-CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR-CYP interactions and underlying conformational changes, improving our understanding of this complex machinery Cytochrome P450 reductase; CPR; conformational; dynamicsrelevant for drug metabolism.
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Project description:Physiological plasticity in a polluted system: A case of an uncultured bacterium and its cypome
| PRJEB38781 | ENA
Project description:The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.
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