• Journal of Microbiology and Biotechnology
• /
• v.20 no.5
• /
• pp.875-880
• /
• 2010

Bacterial enoyl-ACP reductase (FabI) has been demonstrated to be a novel antibacterial target. In the course of our screening for FabI inhibitors, we isolated two methyl-branched fatty acids from Streptomyces sp. A251. They were identified as 14-methyl-9(Z)-pentadecenoic acid and 15-methyl-9(Z)-hexadecenoic acid by MS and NMR spectral data. These compounds inhibited Staphylococcus aureus FabI with $IC_{50}$ values of 16.0 and $16.3\;{\mu}M$, respectively, but did not affect FabK, an enoyl-ACP reductase of Streptococcus pneumonia, at $100\;{\mu}M$. Consistent with their selective inhibition for FabI, they blocked intracellular fatty acid synthesis as well as the growth of S. aureus, but did not inhibit the growth of S. pneumonia. Additionally, these compounds showed reduced antibacterial activity against fabI-overexpressing S. aureus, compared with the wild-type strain. These results demonstrate that the methylbranched fatty acids show antibacterial activity by inhibiting FabI in vivo.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.2
• /
• pp.184-188
• /
• 2013

In the continued search for inhibitors of enoyl-acyl carrier protein (ACP) reductase, we found that four acylbenzenediol sulfate metabolites from Streptomyces sp. AN1761 potently inhibited bacterial enoyl-ACP reductases of Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. Their structures were identified as panosialins A, B, wA, and wB by MS and NMR data. They showed stronger inhibition against S. aureus FabI and S. pneumoniae FabK with $IC_{50}$ of 3-5 ${\mu}M$ than M. tuberculosis InhA with $IC_{50}$ of 9-12 ${\mu}M$. They also exhibited a stronger antibacterial spectrum on S. aureus and S. pneumoniae than M. tuberculosis. In addition, the higher inhibitory activity of panosialin wB than panosialin B on fatty acid biosynthesis was consistent with that on bacterial growth, suggesting that they could exert their antibacterial activity by inhibiting fatty acid synthesis.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.2
• /
• pp.162-173
• /
• 2015

The cellular fatty acid composition is important for metabolic plasticity in Rhodobacter sphaeroides. We explored the effects of changing the cellular ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) in R. sphaeroides by overexpressing several key fatty acid biosynthetic enzymes through the use of expression plasmid pRK415. Bacteria containing the plasmid pRKfabI₁ with the fabI₁ gene that encodes enoyl-acyl carrier protein (ACP) reductase showed a reduction in the cellular UFA to SFA ratio from 4 (80% UFA) to 2 (65% UFA) and had decreased membrane fluidity and reduced cell growth. Additionally, the ratio of UFA to SFA of the chromatophore vesicles from pRKfabI₁-containing cells was similarly lowered, and the cell had decreased levels of light-harvesting complexes, but no change in intracytoplasmic membrane (ICM) content or photosynthetic (PS) gene expression. Both inhibition of enoyl-ACP reductase with diazaborine and addition of exogenous UFA restored membrane fluidity, cell growth, and the UFA to SFA ratio to wild-type levels in this strain. R. sphaeroides containing the pRKfabB plasmid with the fabB gene that encodes the enzyme β-ketoacyl-ACP synthase I exhibited an increased UFA to SFA ratio from 4 (80% UFA) to 9 (90% UFA), but showed no change in membrane fluidity or growth rate relative to control cells. Thus, membrane fluidity in R. sphaeroides remains fairly unchanged when membrane UFA levels are between 80% and 90%, whereas membrane fluidity, cell growth, and cellular composition are affected when UFA levels are below 80%.

• Bulletin of the Korean Chemical Society
• /
• v.32 no.1
• /
• pp.162-168
• /
• 2011

Many ligands have been experimentally designed and tested for their activities as inhibitors against bacterial enoyl-ACP reductase (FabI), ENR. Here the binding energies of the reported ligands with the E. coli ENR-$NAD^+$ were calculated, analyzed and compared, and their molecular dynamics (MD) simulation study was performed. IDN, ZAM and AYM ligands were calculated to have larger binding energies than TCL and IDN has the largest binding energy among the considered ligands (TCL, S54, E26, ZAM, AYM and IDN). The contribution of residues to the ligand binding energy is larger in E. coli ENR-NAD+-IDN than in E. coli ENR-$NAD^+$-TCL, while the contribution of $NAD^+$ is smaller for IDN than for TCL. The large-size ligands having considerable interactions with residues and $NAD^+$ have many effective functional groups such as aromatic $\pi$ rings, acidic hydroxyl groups, and polarizable amide carbonyl groups in common. The cation-$\pi$ interactions have large binding energies, positively charged residues strongly interact with polarisable amide carbonyl group, and the acidic phenoxyl group has strong H-bond interactions. The residues which have strong interactions with the ligands in common are Y146, Y156, M159 and K163. This study of the reported inhibitor candidates is expected to assist the design of feasible ENR inhibitors.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.36 no.3
• /
• pp.237-242
• /
• 2016

Copper (Cu) is a necessary microelement for plants. However, high concentrations of Cu are toxic to plants that change the regulation of several stress-induced proteins. In this study, an annealing control primer (ACP) based approach was used to identify differentially expressed Cu-induced genes in alfalfa leaves. Two-week-old alfalfa plants (Medicago sativa L.) were exposed to Cu for 6 h. Total RNAs were isolated from treated and control leaves followed by ACP-based PCR technique. Using GeneFishing ACPs, we obtained several genes those expression levels were induced by Cu. Finally, we identified several genes including UDP-glucuronic acid decarboxylase, transmembrane protein, small heat shock protein, C-type cytochrome biogenesis protein, mitochondrial 2-oxoglutarate, and trans-2,3-enoyl-CoA reductase in alfalfa leaves. These identified genes have putative functions in cellular processes such as cell wall structural rearrangements, transduction, stress tolerance, heme transport, carbon and nitrogen assimilation, and lipid biosynthesis. Response of Cu-induced genes and their identification in alfalfa would be useful for molecular breeding to improve alfalfa with tolerance to heavy metals.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.35 no.3
• /
• pp.264-268
• /
• 2015

The present research investigated copper and cadmium stress-induced differentially expressed genes (DEGs) using annealing control primers (ACP) with the differential display reverse transcription polymerase chain reaction technique in alfalfa (Medicago sativa L. cv. Vernal) leaves. Alfalfa leaves were subjected to $250{\mu}M$ of copper and cadmium treatment for a period of 6 h. A total of 120 ACPs was used. During copper and cadmium treatment, 6 DEGs were found to be up or down regulated. During copper stress treatment, 1 DEG was up-regulated, and 3 novel genes were discovered. Similarly, during cadmium stress treatment, 1 DEG was up-regulated and 5 novel genes were identified. Among all 6 DEGs, DEG-4 was identified as the gene for trans-2,3-enoyl-CoA reductase, DEG-5 was identified as the gene for senescence-associated protein DIN1 and DEG-6 was identified for caffeic acid O-methyltransferase. All the up-regulated genes may play a role in copper and cadmium stress tolerance in alfalfa.

• Biomedical Science Letters
• /
• v.11 no.4
• /
• pp.455-463
• /
• 2005

Resistance to isoniazid (INH), which is one of the most important drugs in Mycobacterium tuberculosis chemotherapy, has been associated with mutations in genes encoding the mycobacterial catalse-peroxidase (katG), the enoyl acyl carrier protein (ACP) reductase (inhA), alkyl hydroperoxide reductase (ahpC), beta-ketoacyl acyl carrier protein synthase (kasA), and NADH dehydrogenase (ndh). In this study, we examined INH-resistance related genes in 50 INH-resistant and 24 INH-susceptible isolates by PCR-sequence analysis. In brief, mutations at the katG gene were found at codon 315 alone (2/50), at codon 463 alone (19/50), and both at 315 and 463 (29/50). However, while mutations at codon 315 were only detected in INH-resistant isolates, mutations at codon 463 were also detected in INH-susceptible isolates indicating mutations at 463 alone do not seem to confer resistance to INH. Similar to the case of katG 463, some of inhA mutations were also found among INH-susceptible isolates. For example, whereas mutations at 8 upstream of the start codon (UPS) and 15 UPS of the inhA gene were detected only in INH-resistant isolates, mutations at 101, 115, and 125 UPS were detected only in INH-susceptible isolates. Many different kinds of mutations were detected in INH­resistant isolates at ahpC, oxyR gene, and intergenic region of the oxyR-ahpC genes. Howerver, the mutations were not found oxyR and the intergenic regions in INH-susceptible isolates. No mutations were found at either kasA or at ndh gene among INH-resistant isolates. In conclusion, some of mutations such as katG 315, inhA promotor region, and oxyR-ahpC seem to be strongly related to INH-resistance. Currently we are developing a molecular diagnostic method based on these results.