Renaturation of Recombinant ErmSF and Its Refolding Behavior

진형종,Hak Jin Lee 한국분자세포생물학회 2003 Molecules and cells Vol.16 No.2

ErmSF N-말단 부위와 기질인 23S rRNA domain V 중 stem 73-75와의 상호작용 연구

진형종 수원대학교 기능성생명소재연구소 2004 자연과학연구논문집 Vol.3 No.1

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 erm proteins. One of Erm protein 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 25% arginine residues (15/63 amino acids). To find out the interaction mode between N-terminal end region and substrate 23S rRNA, we cloned the DNA fragment coding N-terminal end region polypeptide that NTER(N-terminal end region)1-49, 1-39 and 26-71 by PCR, overexpressed, purified. Using the EMSA(electrophoretic gel mobility shift assay) analysis, we demonstrated that NTER1-49 and NTER1-39 specific interact with the stem 73 and the NTER 26-71 peptides specific interact with the part of stem 75 of 23S rRNA domain V.

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.

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-streptomycin B)계열 항생제 내성인자 단백질인 ErmSF의 N-terminal end loop region에 대한 기능분석 연구

진형종 수원대학교 자연과학연구소 2000 자연과학논문집 Vol.3 No.-

The elm methyltransferases confer resistance to the MLS(macrolide-lincosamide- streptfgramin 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. Several research groups tried to elucidate the protein-RHA interaction but experienceing the severe difficulties because very weak binding affinity between the protein and RNA. But 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), so we have exploited these characteristics to overcome these difficulties. To find out the functional role of N-terminal end region, N-terminal end truncated ermSF was cloned by PCR, expressed as soluble protein in Escherichia coli, and purified with metal(Ni2+) chelation chromatography to yield 59㎎ per liter culture. E. coli cells expressing the truncated ErmSF was found to be resistant to erythromycin. In in vitro, the truncated ErmSF could transfer the methyl group from SAM(S-adenosyl-L-methionine) to the various truncated 23S rRNA substrates (DV, 243, 66, 52, 38 and two 27nt RNAs) but it has retained only 30% activity compared to the wild type ErmSF. 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(71 amino acids)by PCR, overexpressed, purified in the identical manner described above and yielded 7㎎/L. NMR(nuclear mabnetic resonance) study indicated that this polypeptide do not take any secondary structure like α-helix or β-strand, it assumes loop comformation. Using the PACE (polyacrylamide gel coelectrophoresis) analysis, we demonstrated that the polypeptide interact with the 66 nucleotide truncated rRNA with the Kd,app value of 28.3nM. In order to confirm the more derailed interaction mode between N-terminal end region and 235 rRNA, we are carrying out PACE analysis with several truncated rRNA and preparing smaller N-terminal end region peptides.

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.

MLS(macroride-lincosamide-streptograminB) 항생제 내성인자 단백질인 ErmSF에서 높은 보존성을 유지하는 아미노산 -F67-에 대한 기능 연구 : MLS antibiotic resistance factor protein

秦炯淙 水原大學校 2004 論文集 Vol.22 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 erm proteins. ermSF(from Streptomyces fradiae) was cloned, and expressed as soluble protein in E. coli, yielded 126㎎ 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 phenylalanine. These mutant proteins were overexpressed, purified in almost the same amounts as that of wild type ErmSF. Furthermore, these mutant proteins were found to retain native conformation judged by CD (circular dichroism) spectra and observed behaviors during chromatography. In E. coli cells expressing the wild type ErmSF or F67- mutant proteins, F67A, -H. -L, -W and -Y, confer the resistance to erythromycin, but these have quite different enzyme activity in vitro.

ErmSF의 기질인 RNA의 2차 구조가 효소 활동에 주는 영향

秦炯淙 水原大學校 2004 論文集 Vol.22 No.-

Dimethylation by Erm methyltransferases at the N6 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 mechanism. The ErmSF methyltransferase methylates a 665 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. So, we tried to define novel minimal substrate, gradually truncating stem 73 from 72nt RNA which contained all of stem 73 and stem 74. About 20,000 cpm enzyme activity was maintained up to 66nt RNA but 64nt RNA showed about 10% substrate activity. The reduction of substrate activity was found to be due to the dramatic change of the secondary structure of RNA.

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.

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.