Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Sterol 14alpha-demethylase activity in Streptomyces coelicolor A3(2) is associated with an unusual member of the CYP51 gene family.

ABSTRACT: The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various sterol 14alpha-demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated sterols lanosterol and 24-methylene-24,25-dihydrolanosterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanosterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.

SUBMITTER: Lamb DC

PROVIDER: S-EPMC1222601 | biostudies-other | 2002 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Crystal structure of a putative transcriptional regulator SCO0520 from Streptomyces coelicolor A3(2) reveals an unusual dimer among TetR family proteins.

Project description:A structure of the apo-form of the putative transcriptional regulator SCO0520 from Streptomyces coelicolor A3(2) was determined at 1.8 Å resolution. SCO0520 belongs to the TetR family of regulators. In the crystal lattice, the asymmetric unit contains two monomers that form an ?-shaped dimer. The distance between the two DNA-recognition domains is much longer than the corresponding distances in the known structures of other TetR family proteins. In addition, the subunits in the dimer have different conformational states, resulting in different relative positions of the DNA-binding and regulatory domains. Similar conformational modifications are observed in other TetR regulators and result from ligand binding. These studies provide information about the flexibility of SCO0520 molecule and its putative biological function.

| S-EPMC4413006 | biostudies-literature

Streptomyces coelicolor A3(2) SigR regulon

Project description:We determined the genomic locations of the SigR binding sites by chromatin immuno-precipitation of cross-linked DNA and hybridization on a tilling oligonucleotide array after 20 minutes of diamide treatment on cell cultures.

2012-06-03 | GSE33593 | GEO

Sterol 14alpha-demethylase (CYP51) as a therapeutic target for human trypanosomiasis and leishmaniasis.

Project description:Pathogenic protozoa threaten lives of several hundred million people throughout the world and are responsible for large numbers of deaths globally. The parasites are transmitted to humans by insect vectors, more than a hundred of infected mammalian species forming reservoir. With human migrations, HIV-coinfections, and blood bank contamination the diseases are now spreading beyond the endemic tropical countries, being found in all parts of the world including the USA, Canada and Europe. In spite of the widely appreciated magnitude of this health problem, current treatment for sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi) and leishmaniasis (Leishmania spp.) remains unsatisfactory. The drugs are decades old, their efficacy and safety profiles are unacceptable. This review describes sterol 14α-demethylase, an essential enzyme in sterol biosynthesis in eukaryotes and clinical target for antifungal azoles, as a promising target for antiprotozoan chemotherapy. While several antifungal azoles have been proven active against Trypanosomatidae and are under consideration as antiprotozoan agents, crystal structures of sterol 14α-demethylases from three protozoan pathogens, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum provide the basis for the development of new, highly potent and pathogen-specific drugs with rationally optimized pharmacological properties.

| S-EPMC3391166 | biostudies-literature

Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism.

Project description:Streptomyces are filamentous soil bacteria that produce more than half of the known microbial antibiotics. We present the first genome-scale metabolic model of a representative of this group--Streptomyces coelicolor A3(2). The metabolism reconstruction was based on annotated genes, physiological and biochemical information. The stoichiometric model includes 819 biochemical conversions and 152 transport reactions, accounting for a total of 971 reactions. Of the reactions in the network, 700 are unique, while the rest are iso-reactions. The network comprises 500 metabolites. A total of 711 open reading frames (ORFs) were included in the model, which corresponds to 13% of the ORFs with assigned function in the S. coelicolor A3(2) genome. In a comparative analysis with the Streptomyces avermitilis genome, we showed that the metabolic genes are highly conserved between these species and therefore the model is suitable for use with other Streptomycetes. Flux balance analysis was applied for studies of the reconstructed metabolic network and to assess its metabolic capabilities for growth and polyketides production. The model predictions of wild-type and mutants' growth on different carbon and nitrogen sources agreed with the experimental data in most cases. We estimated the impact of each reaction knockout on the growth of the in silico strain on 62 carbon sources and two nitrogen sources, thereby identifying the "core" of the essential reactions. We also illustrated how reconstruction of a metabolic network at the genome level can be used to fill gaps in genome annotation.

| S-EPMC1142472 | biostudies-literature

X-ray structure of 4,4'-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha-demethylase (CYP51).

Project description:A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14alpha-methyl group from sterol precursors by sterol 14alpha-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95A) the structure of CYP51 from Mycobacterium tuberculosis (CYP51(Mt)) co-crystallized with 4,4'-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51(Mt). The newly determined CYP51(Mt)-DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51(Mt)-estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.

| S-EPMC2397474 | biostudies-literature

Indomethacin amides as a novel molecular scaffold for targeting Trypanosoma cruzi sterol 14alpha-demethylase.

Project description:Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have shown recently that specific inhibition of TC sterol 14alpha-demethylase (TCCYP51) with imidazole derivatives is effective in killing both extracellular and intracellular human stages of TC. An alternative set of TCCYP51 inhibitors has been identified using optical high throughput screening followed by web-database search for similar structures. The best TCCYP51 inhibitor from this search was found to have structural similarity to a class of cyclooxygenase-2-selective inhibitors, the indomethacin-amides. A number of indomethacin-amides were found to bind to TCCYP51, inhibit its activity in vitro, and produce strong antiparasitic effects in the cultured TC cells. Analysis of TC sterol composition indicated that the mode of action of the compounds is by inhibition of sterol biosynthesis in the parasite.

| S-EPMC2744100 | biostudies-literature

Structural insights into inhibition of sterol 14alpha-demethylase in the human pathogen Trypanosoma cruzi.

Project description:Trypanosoma cruzi causes Chagas disease (American trypanosomiasis), which threatens the lives of millions of people and remains incurable in its chronic stage. The antifungal drug posaconazole that blocks sterol biosynthesis in the parasite is the only compound entering clinical trials for the chronic form of this infection. Crystal structures of the drug target enzyme, Trypanosoma cruzi sterol 14alpha-demethylase (CYP51), complexed with posaconazole, another antifungal agent fluconazole and an experimental inhibitor, (R)-4'-chloro-N-(1-(2,4-dichlorophenyl)-2-(1H-imid-azol-1-yl)ethyl)biphenyl-4-carboxamide (VNF), allow prediction of important chemical features that enhance the drug potencies. Combined with comparative analysis of inhibitor binding parameters, influence on the catalytic activity of the trypanosomal enzyme and its human counterpart, and their cellular effects at different stages of the Trypanosoma cruzi life cycle, the structural data provide a molecular background to CYP51 inhibition and azole resistance and enlighten the path for directed design of new, more potent and selective drugs to develop an efficient treatment for Chagas disease.

| S-EPMC2919122 | biostudies-literature

Characterization of a novel intracellular endopeptidase of the alpha/beta hydrolase family from Streptomyces coelicolor A3(2).

Project description:In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsin-like activity, absent from the wild type, was detected. Complementation that restored proteasome function did not suppress expression of the endopeptidase. Since the enzyme was not found in two other S. coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant. Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S. coelicolor A3(2) genome with no known homologue. Based on the prediction of a Ser-Asp-His catalytic triad and an alpha/beta hydrolase fold, SCO7095 was assigned to peptidase clan SC. N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization. Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected. On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.

| S-EPMC145308 | biostudies-literature

Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis.

Project description:Sterol 14alpha-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14alpha-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H(37)Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14alpha-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14alpha-demethylases. It catalyzes 14alpha-demethylation of lanosterol, 24, 25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and (1)H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14alpha-methyl group and Delta(8(9))-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14alpha-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.

| S-EPMC17711 | biostudies-other

Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor A3(2).

Project description:Cytochrome P450 170A1 (CYP170A1) is encoded by the sco5223 gene of the Gram-positive, soil-dwelling bacterium Streptomyces coelicolor A3(2) as part of a two-gene cluster with the sco5222 gene. The SCO5222 protein is a sesquiterpene synthase that catalyzes the cyclization of farnesyl diphosphate to the novel tricyclic hydrocarbon, epi-isozizaene (Lin, X., Hopson, R., and Cane, D. E. (2006) J. Am. Chem. Soc. 128, 6022-6023). The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wild-type strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.

| S-EPMC2276382 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data