61 research outputs found
Investigation of Indazole Unbinding Pathways in CYP2E1 by Molecular Dynamics Simulations
Human microsomal cytochrome P450 2E1 (CYP2E1) can oxidize not only low molecular weight xenobiotic compounds such as ethanol, but also many endogenous fatty acids. The crystal structure of CYP2E1 in complex with indazole reveals that the active site is deeply buried into the protein center. Thus, the unbinding pathways and associated unbinding mechanisms remain elusive. In this study, random acceleration molecular dynamics simulations combined with steered molecular dynamics and potential of mean force calculations were performed to identify the possible unbinding pathways in CYP2E1. The results show that channel 2c and 2a are most likely the unbinding channels of CYP2E1. The former channel is located between helices G and I and the B-C loop, and the latter resides between the region formed by the F-G loop, the B-C loop and the β1 sheet. Phe298 and Phe478 act as the gate keeper during indazole unbinding along channel 2c and 2a, respectively. Previous site-directed mutagenesis experiments also supported these findings
Evolutionary origins of the estrogen signaling system : insights from amphioxus
Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Journal of Steroid Biochemistry and Molecular Biology 127 (2011): 176–188, doi:10.1016/j.jsbmb.2011.03.022.Classically, the estrogen signaling system has two core components: cytochrome P450
aromatase (CYP19), the enzyme complex that catalyzes the rate limiting step in estrogen
biosynthesis; and estrogen receptors (ERs), ligand activated transcription factors that interact
with the regulatory region of target genes to mediate the biological effects of estrogen. While the
importance of estrogens for regulation of reproduction, development and physiology has been
well-documented in gnathostome vertebrates, the evolutionary origins of estrogen as a hormone
are still unclear. As invertebrates within the phylum Chordata, cephalochordates (e.g. the
amphioxus of the genus Branchiostoma) are among the closest invertebrate relatives of the
vertebrates and can provide critical insight into the evolution of vertebrate-specific molecules
and pathways. To address this question, this paper briefly reviews relevant earlier studies that
help to illuminate the history of the aromatase and ER genes, with a particular emphasis on
insights from amphioxus and other invertebrates. We then present new analyses of amphioxus
aromatase and ER sequence and function, including an in silico model of the amphioxus
aromatase protein, and CYP19 gene analysis. CYP19 shares a conserved gene structure with
vertebrates (9 coding exons) and moderate sequence conservation (40% amino acid identity with
human CYP19). Modeling of the amphioxus aromatase substrate binding site and simulated
docking of androstenedione in comparison to the human aromatase shows that the substrate
binding site is conserved and predicts that androstenedione could be a substrate for amphioxus
CYP19. The amphioxus ER is structurally similar to vertebrate ERs, but differs in sequence and
key residues of the ligand binding domain. Consistent with results from other laboratories,
amphioxus ER did not bind radiolabeled estradiol, nor did it modulate gene expression on an estrogen-responsive element (ERE) in the presence 59 of estradiol, 4-hydroxytamoxifen,
diethylstilbestrol, bisphenol A or genistein. Interestingly, it has been shown that a related gene,
the amphioxus “steroid receptor” (SR), can be activated by estrogens and that amphioxus ER can
repress this activation. CYP19, ER and SR are all primarily expressed in gonadal tissue,
suggesting an ancient paracrine/autocrinesignaling role, but it is not yet known how their
expression is regulated and, if estrogen is actually synthesized in amphioxus, whether it has a
role in mediating any biological effects . Functional studies are clearly needed to link emerging
bioinformatics and in vitro molecular biology results with organismal physiology to develop an
understanding of the evolution of estrogen signaling.Supported by grants from the NIEHS P42 ES07381 (GVC, SV) and EPA (STAR-RD831301)
(GVC), a Ruth L Kirschstein National Research Service Award (AT, F32 ES013092-01), an NIH
traineeship (SS, SG), a NATO Fellowship (AN) and the Boston University Undergraduate
Research Program (LC)
On the Mechanism of the Oxidation of Toluenes in Artificial P450 Model Systems: Formation of Benzyl Alcohols, Benzaldehydes, and Phenols
Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidogenic cells.
Do mammalian cytochrome P450s exhibit multiple ligand access pathways and ligand channelling?
Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membrane-bound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles
Do mammalian cytochrome P450s show multiple ligand access pathways and ligand channelling?
Comparison of the dynamics of substrate access channels in three cytochrome P450s reveals different opening mechanisms and a novel functional role for a buried arginine
Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity
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