• KSBB Journal
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• v.24 no.3
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• pp.279-286
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• 2009

Streptomyces is well known for their ability to synthesize enormous varieties of antibiotics as secondary metabolites. Among them, S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. However, S. avermitilis also produces oligomycin, which is a potential toxic inhibitor of oxidative phosphorylation in mammalian cells. Therefore, we decided to disrupt oligomycin synthetase gene to prevent co-production of oligomycin in S. avermitilis. To create plasmid for disruption, the smallest gene of oligomycin synthetase gene cluster was obtained by PCR from S. avermitilis chromosome. Then, apramycin resistance gene was inserted in oligomycin synthetase gene for selection. After transformation of this plasmid, oligomycin synthetase gene (olmA5) in the chromosome was displaced with disruption cassette on the plasmid via homologous recombination. As a result of this gene replacement, we obtained mutants (olmA5::apra) that no longer makes the toxic oligomycin. And the mutants confirmed by PCR and HPLC analysis. However, showed no increasement of avermectin production in the mutant was observed.

• Molecules and Cells
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• v.39 no.3
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• pp.211-216
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• 2016

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of $2.6{\AA}$. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant's catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.170-177
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• 1995

To search and develop the compounds exhibiting antifungal activities against the mycelial phase of Candida albicans, approximately 2,900 microorganisms isolated from soil were examined for antifungal activity. Among them, a strain with preferential activity against the mycelial phase of Candida albicans was isolated and identified as Streptomyces sp. A393. Isolation and purification of compounds A393 showing antifungal activity against the mycelial phase of C. albicans were performed using XAD-7 column chromatography, silica gel chromatography, preparative thin- layer silica gel chromatography, and HPLC. The molecular weights of compounds isolated from Streptomyces sp. A393 were determined as 774, 790, 804 and 820. These compounds appeared to have a structure of macrolide antibiotics, oligomycin A, B, C and E. Especially, oligomycin E, which is formerly reported to have no antifungal activity, showed antifungal activity against the mycelial phase of Candida albicans.

• Proceedings of the KSCN Conference
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• 1998.11a
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• pp.91-91
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• 1998

• Preventive Nutrition and Food Science
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• v.5 no.4
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• pp.230-233
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• 2000

The present work was designed to determine whether change in fluidity of the mitochondrial membrane affects mitochondrial {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase characteristics in NIDDM-prone BHE/Cdb rat. Isolated mitochondria fom BHE/Cdb rat fed a 6% coconut oil or corn oil were functionally tested by an analysis of its respiration and the coupling of this process to ATP synthesis in presence of oligomycin, a specific inhibitor of oxidative phosphorylation (OXPHOS), that binds to the {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase. Mitochondria from rats fed coconut oil were more responsive to the inhibitory action of oligomycin with respect to state 3 respiration, respiratory control (RC) ratio and ADP:P (P/O) ratio than were mitochondria from rats fed corn oil. In state 3 respiration, mitochondria from rats fed coconut oil consumed less oxygen than did mitochondria from rats fed corn oil. RC ratio was lower in the mitochondria from rats fed coconut oil than was mitochondria from rats fed corn oil. In P/O ratio, the mitochondria from rats fed coconut oil had a lower P/O ratio than did mitochondria from rats fed corn oil. The data showed that the chang influidity of the mitochondrial membrane by dietary fat affected mitochondrial {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase characteristics. The present study on diet differences in {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase characteristics provides considerable insight into the role diets play in the control of mitochondrial function, expecially OXPHOS in NIDDM with mitochondrial defects.

• The Korean Journal of Physiology
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• v.8 no.2
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• pp.59-66
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• 1974

In recent years much interesting information about the mechanism of the $Na^+-K^+$ activated ATPase has been obtained from investigation of the $K^+-activated$ phosphatase activity which appears to be catalysed by the same enzyme. Also several studies have indicated that a $K^+-activated p-nitrophenylphosphatase activity is intimately related to the ATPase activity. And then the exact relation of p-nitrophenylphosphatase activity to $Na^+-K^+$ ATPase activity is not known. The effects of some ions and drugs on the p-nitrophenylphosphatase activity of the rat brain were investigated and the results were summarized as follows. 1. The p-nitrophenylphosphatase was stimulated markedly by low concentrations of $K^+$, while the activity was activated slightly in the presence of $Na^+$ and oligomycin. 2. Addition of both ATP and $Na^+$ caused a remarkable increase in the activity of the $K^+-dependent$ phosphatase at low concentrations of $K^+$. 3. In the presence of $Na^+$ and low concentrations of $K^+$, oligomycin activated the p-nitrophenylphosphatase. 4. O1igomycin inhibited the stimulation of the enzyme activity caused by $Na^{+}+ATP$. 5. Ouabain inhibited the $K^+-dependent$ p-nitrophenylphosphatase activity more in the presence of ATP and $Na^+$ than in their absence. 6. Quinidine inhibited both $Na^+-K^+$ ATPase and p-nitrophenylphosphatase. These inhibitory effects of the drug were partially antagonized by increasing $K^+$ concentrations. The sensitivity of the $K^+-dependent$ p-nitrophenylphosphatase to quinidine was greater than the that of $Na^+-K^+$ ATPase.

• The Korean Journal of Mycology
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• v.15 no.4
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• pp.224-230
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• 1987

Effects Of organic compound, photosensitizer and $K^+$ ion influx. On the light-induced ATPase of mitochondria in L. edodes purified by linear sucrose density gradient centrifugation were studied. The mitochondrial ATPase activity was investigated by various wavelength illumination at dark state. The mitochondrial ATPase was activated 139% and 128% by 10m mol dithiothreitol and 0.1m mol quinacrine, respectively. This enzyme also was activated 36% by 0.1m mol phenazine methosulfate as photosensitizer. But, 100 mg oligomycin and 1m mol phlorizin inhibited activity of enzyme to 48% and 45%, respectively. Its optimum wavelength was 690 nm on the effect of $K^+$ ion influx, its optimum pH and temperature were found to be 7.2 and $55^{\circ}C$.

• The Korean Journal of Mycology
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• v.17 no.2
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• pp.99-104
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• 1989

Effects of organic compounds, photosensitizers and influx of metal ions on the light-induced mitochondrial ATP synthase in Lentinus edodes purified by stepped sucrose density gradient centrifugation were studied. In our previous work, the activation wavelength and the illumination time of mitochondrial ATP synthase were 470 nm and 15 sec, respectively. This enzyme was activated 85% by 1 mmole 2,6-dichlorophenol indopheol and inhibited by 1 mmole 2,-4-dinitrophenol, $10\;{\mu}mole$ 2-heptyl-4-hydroxyquinoline-N-oxide and $100\;{\mu}g$ oligomycin per ml of ethanol. Particularly, the enzyme was activated 414% by 10 mmole phenazine methosulfate as photosensitizer at 470 nm light. In the influx effects of $Fe^{3+}$ and $Fe^{2+}$ ion, the activity of the above enzyme increased under the optimal light condition compared with nonillumination state.

• Molecules and Cells
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• v.23 no.3
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• pp.363-369
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• 2007

Mitochondria play a central role in energy-generating processes and may be involved in the regulation of channels and receptors. Here we investigated TRPM7, an ion channel and functional kinase, and its regulation by mitochondria. Proton ionophores such as CCCP elicited a rapid decrease in outward TRPM7 whole-cell currents but a slight increase in inward currents with pipette solutions containing no MgATP. With pipette solutions containing 3 mM MgATP, however, CCCP increased both outward and inward TRPM7 currents. This effect was reproducible and fully reversible, and repeated application of CCCP yielded similar decreases in current amplitude. Oligomycin, an inhibitor of $F_1/F_O$-ATP synthase, inhibited outward whole-cell currents but did not affect inward currents. The respiratory chain complex I inhibitor, rotenone, and complex III inhibitor, antimycin A, were without effect as were kaempferol, an activator of the mitochondrial $Ca^{2+}$ uniporter, and ruthenium red, an inhibitor of the mitochondrial $Ca^{2+}$ uniporter. These results suggest that the inner membrane potential (as regulated by proton ionophores) and the $F_1/F_O$-ATP synthase of mitochondria are important in regulating TRPM7 channels.

• 한국유가공학회:학술대회논문집
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• 1997.05a
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• pp.23-34
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• 1997

Calcium-stimulated ATPase ($Ca^{2+}$-ATPase) which has optimal pH value at 7.0 was found in the membrane fraction of human milk, and its enzymatic properties were studied. The purified $Ca^{2+}$-ATPase required 0.45 mM Ca ion for maximal activity. Among the nucleosides, $Ca^{2+}$-ATPase showed a higher substrate specificity to ATP and UTP than to CTP and GTP. $Ca^{2+}$-ATPase had apparent Km value of 0.065, and V max of 7.63 mol ATP hydrolyzed/mg pro-tein per min, respectively. $Ca^{2+}$-ATPase was potently inhibited by lanthanide, vanadate, and p-chloromercuribenzoate, and inactivated by EDTA, and CDTA and EGTA, but were unaffected by N-ethylmaleimide, $NaN_3$, ouabain, or oligomycin, and was completely inactivated by heating at $60^{\circ}C$ for 10 min. This enzyme activity was concentrated in the membrane fraction of the cream and skim milk membrane, but not founded in bovine milk.