ErmSF 23S rRNA methyltransferase에서 높은 보존성을 유지하는 아미노산 64S와 65Q에 대한 기능 연구

秦炯淙 水原大學校 2000 論文集 Vol.18 No.-

Microorganisms including pathogens predominantly exhibit the resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics by dimethylation of the single adenine residue of 23S rRNA. This chemical modification is mediated by a group of enzymes named elm proteins. ermSF (from Streptomycees fradiae)was cloned, and expressed as soluble protein in E. coli, yielded 126mg per liter of culture. Motif, -64SQNF67- of ErmSF is conserved in almost all members of erm family proteins. To find out its functional role, we used site-directed mutagenesis to replace glutamine and serine. These mutant proteins were overexpressed, purified in the identical manner described above and yielded almost same amounts of proteins. Furthermore, these mutant proteins were found to retain native comformation judged by CD(circular dichroism) spectra and observed behaviours during chromatography. In E. coli cell, Q65 mutant proteins, Q65E, -H, -N and -R did not confer the resistance to erythromycin while E. coli cells expressing the wild type ErmSF were resistant to erythromycin. Even in vitro, all of the purified Q65 mutant proteins could not transfer the methyl group from SAM(S-adenosyl-L-methionine) to the various truncated 235 rRNA substrates(BV, 432, 243, 66, 52, 41, 38 and 27nt RNA). In case of S64 mutant proteins, different Phenomena were observed. That is, E. coli cells expressing S64A, -C, -G mutant proteins were resistant to erythromycin but S64F, -T, -Y mutant proteins which have longer side-chain than serine did not confer the resistant to erythromycin. Recently, Schluckebier G. and coworkers(J. Mol. Biol., 1999) reported that highly conserved Asn next to Gln 65 forms hydrogen bond with the positively charged Sδ of SAM through the main chain oxygen. Therefore these results suggested that 1) residue 65 of ErmSF strictly requires glutamine for enzyme activity, 2) probably gln 65 directly involves in methyl group transfer reaction or interacts with nucleotide of 23S rRNA near the methylatable adenine to position the A2058 to the active center. 3) Residue 64 of ErmSF does not strictly requires serine for enzyme activity but it requires small side-chain amino acid which does not provoke steric hindrance in 'binding pocket' where methylatable adenine residue of 23S rRNA and cofactor SAM are located close to each other.

제거 변이체를 이용한 ErmSF 내의 Motif 60RRE62의 기능 분석

진형종 수원대학교 기능성생명소재연구소 2007 자연과학연구논문집 Vol.6 No.1

The erm methyltransferases confer resistance to the MLS (macrolide-lincosamide- streptogramin B) antibiotics by mono-, or dimethylation of sin히e adenine residue within bacterial 23S rRNA. This chemical modification is mediated by a group of enzymes named erm proteins. One of Erm protein ErmSF, from Streptmyces fradiae, recognizes motifs within domain V of the rRNA that specifically targets adenine 2058 (A2058 in E. coli numbering) for methylation. Unlike the other homologous proteins, ErmSF contains long N-terminal end region (63 amino acids) and it contains 25% arginine residues (15/63 amino acids). 60RRE62 motif existed in C-terminal end of this long N-terminal end region. We confirmed the function of this motif using mutant proteins which truncated this motif.

ErmSF의 새로운 최소 기질에 대한 연구

진형종 수원대학교 기능성생명소재연구소 2004 자연과학연구논문집 Vol.3 No.1

Dimethylation by Erm methyltransferases at the N-6 position of adenine 2085 (A2085, Bacillus subtilis numbering) in domain V of bacterial 23 S rRNA confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics. The ErmSF, from Streptmyces fradiae, recognizes motifs within domain V of the rRNA that specifically targets adenine 2085 for methylation. Considering the protein structure, the interaction between the protein and the substrate, 23S rRNA is found to be achieved by novel recognition rnechanism. The ErmSF methyltransferase methylates a 605 nucleotide transcript of domain V as efficiently as it methylates intact 23S rRNA. Recently, Douthwaite and his coworker suggested that the minimal substrate RNA transcript that shows methyl-accepting activity is a 27nt stem-loop, corresponding to the stem 73 in the 23S rRNA. But unlike the other homologous proteins, ErmSF contains a long N-terminal end region. This N-terminal end region is important to maintain the ErmSF enzyme activity. ErmSF could methylated the 55nt RNA substrate which contained whole stem74 and shorter stem73 then 27nt RNA.

MLS계열 항생제 내성인자 단백질인 ErmSF의 N-말단 부위와 상호 작용하는 기질 RNA의 탐색

진형종 수원대학교 기능성생명소재연구소 2003 자연과학연구논문집 Vol.2 No.2

The erm methyltransferases confer resistance to the MLS (macrolide-lincosamide- streptogramin B) antibiotics by dimethylation of single adenine residue within bacterial 23S rRNA. The ErmSF, from Streptmyces fradiae, recognizes motifs within domain V of the rRNA that specifically targets adenine 2085 (A2085 in Bacillus subtilis numbering) for methylation. Considering the protein structure, the interaction between the protein and the substrate, 23S rRNA is found to be achieved by novel recognition mechanism. Unlike the other homologous proteins, ErmSF contains long N-terminal end region (63 amino acids) and it contains 25% arginine residues (15/63 amino acids). To find out the interaction mode between N-terminal end region and 23S rRNA, we cloned the DNA fragment coding N-terminal end region polypeptide that NTER(N-terminal end region)l-49 and 26-71 by PCR, overexpressed, purified. Using the EMSA(electrophoretic gel mobility shift assay) analysis, we demonstrated that NTER1-49 and NTER26-71 specific interact with the stem 73-75 of 23s rRNA domain V.

MLS(macroride-lincosamide-streptogramin B)항생제 내성인자 단백질인 ErmSF 내의 Motif-60RRE62-가 기질 RNA에 Mono 또는 Dimethylation시키는 기작에 주는 영향

秦炯淙 水原大學校 2005 論文集 Vol.23 No.-

The erm methyltransferases confer resistance to the MLS (macrolide-lincosamide- streptogramin B) antibiotics by mono-, or dimethylation of single adenine residue within bacterial 23S rRNA. The last decade has seen the isolation and characterization of approximately 30 erm genes from diverse sources, ranging from clinical pathogens to actinomycetes that produce antibiotics. Erm proteins show high degree of amino acid sequence homology and comprise of a group of structurally homologous N-methyltransferase. The ErmSF, from Streptmyces fradiae, recognizes motifs within domain V of the rRNA that specifically targets adenine 2085 (A2085 in Bacillus subtilis numbering) for methylation. Unlike the other homologous proteins, ErmSF contains long N-terminal end region (63 amino acids) and it contains motif -60RRE62- which have been known to interact with GG-C site in DNA. This motif amino acids were mutated through site-directed mutagenesis. Those mutant protein's monometyltransfer activity was maintained but dimetyltransfer activity was decreased.

MLS(macrolide-lincosamide-streptogramin B) 항생제 내성인자인 ErmSF 단백질에 있어서 최소기질의 확립

秦炯綜 水原大學校 2000 論文集 Vol.18 No.-

Erm methyltransferases specifically modify a single adenine residue(A2085 in Bacillus subtilis) within bacterial 23S rRNA conferring resistance to MLS (macrolide -lincosamide-streptogramin B ) antibiotics . The ErmSF methyltransferase from Streptomyces fradiae was expressed and purified as soluble protein in E. coli using immobilized metal ion(Ni2+) affinity chromatography to the yield of 126 mg/L culture. The recombinant ErmSF efficiently methylates in vitro transcribed domain V(623nt) of 23S rRNA as well as whole 23S rRNA. Domain V of 23S rRNA(DV) was reported to have all the elements required for full erm methyltransferase activity [ Jin, H. J., et al. (1994) J. Bacteriol. 176, 6992-6998]. To identify the minimalist substrate, DV was tried to be truncated. We synthesized the various size of RNA fragment from DV such as 432, 243, 88, 66, 52, 41, 38, 27 and another 27 nt RNA fragment and confirmed their methyl group accepting activities. In the process of identifying minimalist substrate, we found out novel 27 nucleotide minimalist substrate containing part of stem 74, unpaired sequence including methylatable adenine, 4 nucleotides of stern 73 and tetraloop sequence UUCG.

Extremophile Bacillus halodurans C-125의 macrolide 계열 항생제 내성 유발 인자 분석

진형종 水原大學校 2012 論文集 Vol.26 No.-

Bacillus halodurans is a rod-shaped, Gram-positive, motile and spore-forming bacterium found in soil. Facultatively alkalifilic Bacillus halodurans C-125 strain encodes an unchracterized erm gene with a mphB gene that confer resistance to macrolide antibiotics in chromosomal DNA. By a BLASTP analysis, the gene erm product has 66.9% identity to the amino acid sequence of ErmK from B. licheniformis, and the gene mphB product has 54.8% identity to MPH(2')Ⅱ encoded by mphB from E. coli. Bacillus halodurans C-125 has an inducible resistance that confirmed by antibiotics susceptibility assay and minimal inhibition concentration (MIC) test.

23S rRNA Methyltransferase인 ErmSF의 효소동력학적 연구

진형종 수원대학교 기능성생명소재연구소 2005 자연과학연구논문집 Vol.4 No.1

ErmSF is a methyltransferase that confers resistance to the macrolide - lincosamide - streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmSF was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Kinetic studies of the monomethylation reaction showed that the apparent K_m and V_max of 72 and 66-nucleotide RNA oligonucleotide was almost same the value determined for domain V of 23S rRNA.

菟絲子 의效能에 대한 硏究

진상식,황의현,김형창,김천수,오광수,한종현 한국전통의학연구소 2002 한국전통의학지 Vol.12 No.1

The purpose of this study is to determine the effect of Semen Cuscutae on phenylephrine(PE) and KCI induced contraction of isolated rat thoracic aorta. Contractile force was measured with force displacement transducer under 1.5 g loading tension. Contraction evoked by PE (ED_(50)) was relaxed significantly by Semen Cuscutae. The mean percent relaxing of PE induced contraction was 60.6% (p<0.01) after 300 ㎕/㎖ Semen Cuscutae, but the mean percent contraction of KCI induced contraction was 10.4% (p<0.05) after 300 ㎕/㎖ Semen Cuscutae. Vasorelaxation by Semen Cuscutae was significantly reduced after with and without endothelial cells. Pretreatment of aortic rings with L-NAME (100 μM), a nitric oxide synthase inhibitor, indomethacin (3 μM), an inhibitor of cyclooxygenase, methylene blue (10 μM), guanylyl cyclase inhibitor, propranolol (1 μM), a β-adrenoceptor antagonist, atropine (1 μM), a muscarinic receptor antagonist and diltiazem (10 μM), a Ca^(2+)-channel blocker was recovered the effect aorta. These results indicate that Semen Cuscutae can relax EP induced contraction of isolated rat thoracic aorta and that this increasing contraction related to epithelium and various effect.

In vito activity 와 kinetic analysis를 통한 ErmSF와 23S rRNA의 interaction mode의 연구

진형종 수원대학교 자연과학연구소 2000 자연과학논문집 Vol.3 No.-

Recently, frequent appearance of bacterial pathogens resistant to various available antibiotics is the most serious problem to the health and life of human being. One of the most plausible strategies to overcome this medical disaster is to develop the inhibitor of antibiotic resistance factor. ErmSF is a methyltransferase that confers resistance to the MLS (macrolide-lincosamide-streptograminB group) antibiotics by catalyzing the mono- or dimethylation of 23S rRNA peptidyltransferase loop at a specific N6-position of adenine residue(A2085 in Bacillus subtilis). The ermSF gene was cloned from Streptomyces fradiae NRRL2702 by PCR method and expressed to a high level in E. coli BL2l(DE3). ErmSF was obtained and purified as a soluble form in the yield of 126㎎/L culture and the enzyme efficiently methylates a 23S rRNA [Jin. H. J.(1999) Mol. Cells ]and also a 623nt transcript of domain V of 23S rRNA in vitro. Domain V of 23S rRNA was reported to have all the elements required for full erm methyltransferase activity [ Jin. H. J., et at. (1994) J. Bacteriol, 176, 6992-69981]. By progressively truncating domain V (DV, 432, 243, 66, 52, 41, 38, 27nt RNA), novel 27nt RNA minimalist substrate for ErmSF was identified, Unlike the other homologous erm proteins, ErmSF contains long N-terminal end region(71 a. a), 25% of which amino acids is composed of arginine known to interact well with RNA. From this characteristic and results of our preliminary experiments, this region is thought to interact with nucleotides of DV stems 74-79. To confirm this notion, antibiotic susceptibility assay, in vitro methylation and kinetic analysis using the wild type and the N-terminal end region truncated ErmSF and various RNAs mentioned above were carried out. Even though it confers resistance to erythromycin in E. coli, in in vitro methylation reaction, N-terminal end region truncated ErmSF showed only 30% activity compared to the wild type ErmSF in vitro. The kinetic studies performed under the conditions that only monomethylation occurs showed that the apparent Km of 243nt RNA(462 nM) and 66nt RNA(3787.5 nM) was 9 fold and 69 fold greater than the value determined for DV(44.43 nM), respectively. In addition, the Vmax for these fragments also increased 1.4 fold(1.15 pmol/min/㎎ ErmSF) and 4.2 fold(3.37 pmol/min/㎎ ErmSF) compared to that of DV(0.84 pmol/min/㎎ ErmSF). In contrast, when these RNA substrates were interacted with N-terminal end region truncated ErmSF, the Km values of DV, 243nt and 66nt RNAs were 851.32 nM, 25281.1 nM and 98096.7 nM. Vmax values were 1.59 pmol/min/㎎ ErmSF, 2.2 pmol/min/㎎ ErmSF and 40.32 pmol/min/㎎ ErmSF. When these values were compared with that obtained using the wild type ErmSF with DV, Km value were increased 17 fold, 52 fold and 2097 fold and Vmax value were also increased 1.9 fold, 2.6 fold and 49 fold. Therefore, these results suggested that N-terminal end region of ErmSF directly interact with seem 74-79 of 23S rRNA and enhance the protein-RNA interaction for methyl group accepting activity.