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The Journal of Biological Chemistry Apr 2000The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose...
The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose moiety from UDP-glucose (UDP-Glc) to many macrolides. The glycosyltransferase coded by the oleD gene has been purified 371-fold from a Streptomyces lividans clone expressing this protein. The reaction product was isolated, and its structure determined by NMR spectroscopy. The kinetic mechanism of the reaction was analyzed using the macrolide antibiotic lankamycin (LK) as substrate. The reaction operates via a compulsory order mechanism. This has been shown by steady-state kinetic studies and by isotopic exchange reactions at equilibrium. LK binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated lankamycin (GS-LK). A pH study of the reaction was performed to determine values for the molecular pK values, suggesting possible amino acid residues involved in the catalytic process.
Topics: Amino Acid Sequence; Anti-Bacterial Agents; Catalysis; Erythromycin; Glucosyltransferases; Glycosylation; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Oleandomycin; Protein Folding; Streptomyces antibioticus; Uridine Diphosphate; Uridine Diphosphate Glucose
PubMed: 10766792
DOI: 10.1074/jbc.275.16.11713 -
European Journal of Biochemistry May 2000The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM-3 (CYP102) were studied using absorption...
The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM-3 (CYP102) were studied using absorption spectroscopy in the ultraviolet and visual regions. Conformational changes in the overall protein structures of both CYP3A4 and CYP102 due to the effects of temperature and pressure are reversible. However, the enzymes differ in the properties of their active sites. The CYP3A4 enzyme denatures to the inactive P420 form relatively easy, at 3000 bar over half is converted to P420. The compressibility of its active site is lower than that of CYP102 and is greater with the substrate bound, which is in line with the observed lack of a stabilizing effect of the substrate on its conformation under pressure. In contrast, CYP102, although having the most compressible active site among the P450s, possesses a structure that does not denature easily to the inactive (P420) form under pressure. In this respect, it resembles the P450 isolated from acidothermophilic archaebacteria [McLean, M.A., Maves, S.A., Weiss, K.E., Krepich, S. & Sligar, S.G. (1998) Biochem. Biophys. Res. Commun. 252, 166-172].
Topics: Bacterial Proteins; Binding Sites; Carbon Monoxide; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Enzyme Stability; Escherichia coli; Heme; Mixed Function Oxygenases; NADPH-Ferrihemoprotein Reductase; Plasmids; Pliability; Pressure; Protein Binding; Protein Conformation; Spectrophotometry; Temperature; Troleandomycin
PubMed: 10806389
DOI: 10.1046/j.1432-1327.2000.01305.x -
Biochemistry Jul 2007Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s...
Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in Saccharomyces cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal (MG-132 and MG-262) and ALD [NH4Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase with immunoprecipitation analyses revealed a basal 52% 35S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH4Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were cross-linked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-cross-linking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.
Topics: Adenine; Adenosine Triphosphatases; Ammonium Chloride; Animals; Autophagy; Boronic Acids; Cell Cycle Proteins; Cross-Linking Reagents; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Dexamethasone; Dicarbethoxydihydrocollidine; Formaldehyde; Hepatocytes; Leupeptins; Lysosomes; Male; Nuclear Proteins; Polymers; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Saccharomyces cerevisiae Proteins; Troleandomycin; Ubiquitin; Valosin Containing Protein
PubMed: 17550236
DOI: 10.1021/bi700340n -
Journal of Bacteriology Aug 1998In the search for heterologous activators for actinorhodin production in Streptomyces lividans, 3.4 kb of DNA from Streptomyces rochei F20 (a streptothricin producer)...
In the search for heterologous activators for actinorhodin production in Streptomyces lividans, 3.4 kb of DNA from Streptomyces rochei F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the actII-ORF4 gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3' end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3' terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named msr, is a new bacterial multidrug ABC transporter.
Topics: ATP-Binding Cassette Transporters; Amino Acid Sequence; Anthraquinones; Anti-Bacterial Agents; Bacterial Proteins; Cloning, Molecular; DNA, Bacterial; Drug Resistance, Microbial; Drug Resistance, Multiple; Genes, Bacterial; Molecular Sequence Data; Open Reading Frames; Sequence Alignment; Sequence Homology, Amino Acid; Streptomyces; Transcriptional Activation
PubMed: 9696745
DOI: 10.1128/JB.180.16.4017-4023.1998 -
Proceedings of the National Academy of... Sep 1985It has not yet been determined whether human liver contains inducible cytochromes P-450 similar to those that catalyze the oxidative metabolism of foreign substances in... (Comparative Study)
Comparative Study
It has not yet been determined whether human liver contains inducible cytochromes P-450 similar to those that catalyze the oxidative metabolism of foreign substances in animals. We carried out immunoblot analyses of liver microsomes isolated from eight patients and found that each contained a cytochrome P-450, termed HLp, that reacted with antibodies directed against P-450p, a rat liver cytochrome that is inducible by the anti-glucocorticoid pregnenolone-16 alpha-carbonitrile, by glucocorticoids, by anti-seizure drugs, and by such macrolide antibiotics as triacetyloleandomycin. In the two patients who received dexamethasone and anti-seizure medications and in the one patient who was given triacetyloleandomycin, the concentrations of immunoreactive HLp and the ability to demethylate erythromycin and/or to convert triacetyloleandomycin to a metabolite that forms a spectral complex with cytochrome P-450 heme (catalytic properties unique to P-450p in rat liver) were significantly higher as compared to the values for patients who received no inducing drugs. We purified HLp to homogeneity and found that it was immunochemically related to P-450p and to its homologue in the rabbit (LM3c), actively demethylated erythromycin in a reconstituted system, exhibited electrophoretic mobility identical to that of P-450p, and shared 57% homology in its NH2-terminal amino acid sequence with that of a pregnenolone-16 alpha-carbonitrile-inducible rat cytochrome P-450. We conclude that HLp is a human representative of the multigene family of the glucocorticoid-inducible cytochromes P-450.
Topics: Amino Acid Sequence; Cytochrome P-450 Enzyme System; Dexamethasone; Enzyme Induction; Erythromycin; Humans; Immunosorbent Techniques; Liver; Substrate Specificity; Troleandomycin
PubMed: 3898085
DOI: 10.1073/pnas.82.18.6310 -
The Journal of Biological Chemistry Feb 1987We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation. The ermC gene product is known to methylate...
We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation. The ermC gene product is known to methylate rRNA, rendering ribosomes unable to bind this antibiotic. We have shown that the induction of ermC methylase in vitro is post-transcriptional and specific for the macrolides erythromycin and oleandomycin. Erythromycin has no significant effect on the stability of the ermC transcript in vitro, and hence the post-transcriptional induction of methylase under these conditions occurs by stimulation of translation. The induction effect requires ribosomes able to bind erythromycin. By adding small proportions of unmethylated to a methylated extract in the presence of erythromycin, methylase synthesis could be induced. Conversely, when small amounts of methylated extracts were mixed with unmethylated extracts, methylase synthesis could be maintained at elevated levels in the presence of a high concentration of erythromycin. These effects were specific for the inducible ermC, were not observed with a constitutive variant, and could be explained satisfactorily by the translational attenuation model. The roles of three segments of the ermC leader in regulation were explored by probing with appropriate complementary synthetic oligodeoxynucleotides. The induction effect of erythromycin was mimicked by using an oligonucleotide that could free the ribosome binding site for methylase.
Topics: Bacillus subtilis; Bacterial Proteins; DNA (Cytosine-5-)-Methyltransferases; Electrophoresis, Polyacrylamide Gel; Magnesium; Nucleic Acid Conformation; Protein Biosynthesis; Protein Sorting Signals; Substrate Specificity
PubMed: 3027098
DOI: No ID Found -
Comparative Medicine Dec 2008The objective of this study was to demonstrate that Bama miniature pigs are a suitable experimental animal model for the evaluation of drugs for man. To this end, in... (Comparative Study)
Comparative Study
The objective of this study was to demonstrate that Bama miniature pigs are a suitable experimental animal model for the evaluation of drugs for man. To this end, in vitro lovastatin metabolism at the minipig liver microsomal level and in vivo pharmacokinetics were studied. Results were compared with those obtained from humans. Our data indicate that the main metabolites and enzyme kinetic parameters of lovastatin metabolism are similar in pigs and humans. Triacetyloleandomycin, a specific inhibitor of human CYP3A4, inhibited the metabolism of lovastatin in pig and human liver microsomes. In addition, the pharmacokinetic parameters and absolute bioavailability suggested that the absorption and elimination of lovastatin in Bama miniature pigs were similar to those in humans. Lovastatin was distributed across many organs in pigs, but the highest levels were found in the stomach, intestines, and liver. Within 96 h, 7% and 82% of the given dose was excreted in the urine and feces, respectively. In addition, no significant species differences in the plasma protein binding ratio of lovastatin and the rates of lovastatin hydrolysis to beta-hydroxyacid lovastatin were apparent. From these results, we conclude that Bama miniature pigs are suitable for use in drug evaluation studies.
Topics: Animals; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Drug Evaluation, Preclinical; Enzyme Inhibitors; Humans; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; In Vitro Techniques; Lovastatin; Male; Microsomes, Liver; Models, Animal; Species Specificity; Swine; Swine, Miniature; Troleandomycin
PubMed: 19149415
DOI: No ID Found -
Clinical Pharmacology and Therapeutics Nov 2002The intravenous (14)C-erythromycin breath test (ERMBT(IV)) does not measure aggregate liver and intestinal cytochrome P450 (CYP) 3A4 activity. Accordingly, we evaluated...
BACKGROUND
The intravenous (14)C-erythromycin breath test (ERMBT(IV)) does not measure aggregate liver and intestinal cytochrome P450 (CYP) 3A4 activity. Accordingly, we evaluated an oral stable-labeled ((13)C) formulation of the test (ERMBT(oral)) as an alternative CYP3A4 phenotyping probe.
METHODS
After an overnight fast, 14 young healthy volunteers (5 women and 9 men) received the ERMBT(IV) (0.07 micromol, 3 muCi), followed by the ERMBT(oral) (500 mg). The next morning, the CYP3A4 inhibitor troleandomycin (500 mg) was given, and both ERMBTs were repeated. After at least 24 hours, the CYP3A4 and P-glycoprotein inducer rifampin (600 mg; INN, rifampicin) was given daily for 7 days, and both ERMBTs were repeated 24 hours after the last dose of rifampin. Plasma samples were collected for 10 hours with each administration of the ERMBT(oral), and erythromycin levels were measured by liquid chromatography-mass spectrometry. Finally, the effect of troleandomycin on erythromycin transport was examined in Caco-2 cell monolayers.
RESULTS
Compared with baseline values, the median ERMBT(IV) and ERMBT(oral) results and erythromycin apparent oral clearance (CL/F) all significantly decreased, by at least 70%, with troleandomycin treatment (P =.001 for each comparison). With rifampin treatment, the median ERMBT(IV) result and CL/F increased 2-fold (P < or =.01), but the median ERMBT(oral) result was unchanged (P =.30). There were no rank-order correlations between the ERMBT(IV) and ERMBT(oral) results or between either ERMBT result and CL/F within each treatment group (P > or =.07). In addition, troleandomycin had no effect on erythromycin transport in Caco-2 cells (P > or =.20).
CONCLUSIONS
The ERMBT(oral) was influenced by processes in addition to intestinal and hepatic CYP3A4 activity and therefore did not provide a straightforward measure of aggregate CYP3A4 phenotype. The erythromycin-rifampin interaction cannot be attributed to CYP3A4 induction alone and probably also reflected intestinal P-glycoprotein induction.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adult; Biological Transport; Breath Tests; Carbon Dioxide; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Interactions; Erythromycin; Female; Humans; Male; Rifampin; Troleandomycin
PubMed: 12426516
DOI: 10.1067/mcp.2002.128387 -
Antimicrobial Agents and Chemotherapy Apr 1988Clinical isolates of Staphylococcus and Arthrobacter spp. were screened for lincosamide resistance. Six different patterns of resistance were found. Strains designated...
Clinical isolates of Staphylococcus and Arthrobacter spp. were screened for lincosamide resistance. Six different patterns of resistance were found. Strains designated SF27 and SF28 showed low-level resistance to lincosamides: one was susceptible to erythromycin (SF27) and the other was resistant (SF28). Analysis of ribosomes from the resistant strains in an in vitro poly(U)-dependent protein-synthesizing system showed that ribosomes of both strains were sensitive to lincomycin and clindamycin. Four patterns of high-level resistance to lincosamides were observed (strains SF4, SF19, SF30, and SF31). All of these except SF30 had ribosomes which were highly resistant in vitro to the antibiotics and showed a close correlation with results of the in vivo experiments. In vivo protein synthesis by strain SF30 was resistant to lincomycin and sensitive to clindamycin, whereas the ribosomes were sensitive when assayed in vitro. Lincosamide-inactivating enzymes were not detected in cell extracts of the six resistant strains. Strains SF19 and SF31 demonstrated two ribosome-mediated lincosamides resistance mechanisms that were not previously reported. Both strains were highly resistant to lincosamides and susceptible to erythromycin, but SF19 was also highly resistant to oleandomycin and partially resistant to various macrolides.
Topics: Aminoglycosides; Anti-Bacterial Agents; Arthrobacter; Bacterial Proteins; Clindamycin; Drug Resistance, Microbial; Erythromycin; Lincomycin; Microbial Sensitivity Tests; Ribosomes; Staphylococcus
PubMed: 3377455
DOI: 10.1128/AAC.32.4.420 -
Antimicrobial Agents and Chemotherapy Mar 1987The distribution of nucleotide sequences related to ereA, ereB, and ermAM was studied by colony hybridization in 112 strains of members of the family Enterobacteriaceae...
The distribution of nucleotide sequences related to ereA, ereB, and ermAM was studied by colony hybridization in 112 strains of members of the family Enterobacteriaceae that are highly resistant to erythromycin. The ereA and ereB genes encoding erythromycin esterases type I and II, respectively, were detected in strains inactivating the 14-membered macrolides erythromycin and oleandomycin. Because all 52 strains resisting these antibiotics by inactivation were detected by ereA (n = 23), ereB (n = 23), or both probes (n = 6), only two classes of genes accounted for this resistance phenotype. The ermAM gene encoding a streptococcal rRNA methylase was detected in 21 strains of Escherichia coli and two strains of Klebsiella spp. Determination of the MICs of macrolide, lincosamide, and streptogramin (MLS) antibiotics demonstrated a correlation between hybridization with ermAM and the so-called MLS resistance phenotype. The presence of 11 strains coresistant to MLS antibiotics that did not hybridize to the ermAM probe suggests that, as in gram-positive organisms, MLS resistance in members of the family Enterobacteriaceae involves more than one class of rRNA methylase. Numerous strains (n = 18) were found to produce both an erythromycin esterase type II and an rRNA methylase. Physical linkage between ereB and ermAM may be responsible for the codissemination of the genes. Despite their exogenous origin, ereB and ermAM are already disseminated in various genera of the Enterobacteriaceae.
Topics: Carboxylic Ester Hydrolases; Drug Resistance, Microbial; Enterobacteriaceae; Erythromycin; Genes, Bacterial; Methyltransferases; Nucleic Acid Hybridization; Phenotype; Plasmids
PubMed: 3579257
DOI: 10.1128/AAC.31.3.404