Deletion of N-terminal End Region of ErmSF Leads to an Amino Acid Having Important Role in Methyl Transfer Reaction

ErmSF에서 특이적으로 발견되는 N-terminal End Region의 점차적인 제거에 의한 활성에 중요한 아미노산의 규명

  • Lee Hak Jin (Department of Genetic Engineering, College of Natural Science, The University of Suwon) ;
  • Jin Hyung Jong (Department of Genetic Engineering, College of Natural Science, The University of Suwon)
  • 이학진 (수원대학교 자연과학대학 생명공학과) ;
  • 진형종 (수원대학교 자연과학대학 생명공학과)
  • Published : 2004.12.01


ErmSF is one of the ERM proteins which transfer the methyl group to A2058 in 23S rRNA to confer the resis­tance to MLS (macrolide-lincosamide-streptogramin B) antibiotics on microorganism. Unlike other ERM pro­teins, ErmSF contains long N-terminal end region (NTER), of which $25\%$ is composed of arginine that is known to interact with RNA well. Gradual deletion of NTER leaded us to the point where mutant protein lost much of activity in vivo. Overexpressed and purified mutant protein showed much reduced activity in vitro: $2\%$ activity relative to that of wild type protein. This fact suggests that this amino acid interact with RNA close to meth­ylatable adenine to locate it at an active site properly.


ErmSF;in vivo activity;in vivo activity;MLS (macrolide-incosamide-streptogramin B) antibiotic resistance factor protein


  1. Buriankova, K., F.D. Populaire, O. Dorson, A. Dondran, J.C. Ghnassia, J. Weiser, and J.L. Nd Pernadet. 2004. Molecular basis of intrinsic macrolideresistance in the Mycobacterium tuberculosis complex. Antimicrob. Agents Chemother. 48, 143-150
  2. Bussiere, D.E., S.W. Muchmore, C.G. Dealwis, G. Schluckebier, V.L. Nienaber, R.P. Edalji, K.A. Walter, U.S. Ladror, T. F. Holzman, C. Abad-Zapatero. 1998 Crystal structure of ErmC', an rRNA methyltransferase which mediates antibiotic resistance in bacteria. Biochemistry. 37, 7103-7112
  3. 'Frontiers in Biotechnology : Antibiotic Resistance' 1994. Science 264, 317-476
  4. Kovalic, D., J.H. Kwak, and B. Weisblum. 1991. General method for direct cloning of DNA fragments generated by the polymerase chain reaction. Nucleic Acid Res. 19, 4650
  5. Skinner, R., E. Cundliffe, and F.J. Schmidt. 1983. Site for Action of a ribosomal RNA methylase responsible for resistance to erythromycin and other antibiotics. J. Biol. Chem. 258, 12702-12706
  6. Weisblum, B. 1995. Erythromycin resistance by ribosome modification. Antimicrob. Agents Chemother. 39, 577-585
  7. Zalacain, M. and E. Cundliffe. 1991. Cloning of tlrD, a fourth resistance gene, from the tylosin producer, Streptomyces fradiae. Gene 97, 137-142
  8. Zalacain, M. and E. Cundliffe. 1989. Methylation of 23S rRNA by tlrA(ermSF), a tylosin resistance determinant from Streptomyces fradiae. J. Bacteriol. 171, 4254-4260
  9. Jin H.J. and Y.D. Yang. 2002. Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli. Protein Expr. Purif. 25, 149-59
  10. Lai, C.J., B. Weisblum, S.R. Fahnestock, and M. Nomura. 1973. Alteration of 23 S ribosomal RNA and erythromycin-induced resisitance to lincomycin and spiramycin in Staphylococcus aureus. J. Mol. Biol. 74, 67-72
  11. 진형종. 2001. MLS (macrolide-lincosamide-streptogramin B) 항생제 내성인자 단백질인 ErmSF domain 발현. Kor. J. Microbiol. 37, 245-252
  12. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature. 227, 680-685
  13. Maravic, G., M. Feder, S. Ponger, M. Fogel, and J.M. Bujnicki. 2003. Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m$^6$A methyltransferase ErmC´. J. Mol. Biol. 332, 99-109
  14. Schluckebier G, Zhong P, Stewart KD, Kavanaugh TJ, Abad-Zapatero C. 1999. The 2.2 $\AA$ structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism. J Mol. Biol. 289, 277- 291
  15. Vester, B., and S. Douthewaite. 1994. Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methytransferase. J. Bacteriol. 176, 6999-7004
  16. Cundliffe, E. 1989. How antibiotic-producing organisms avoid suicide. Annu. Rev. Microbiol. 43, 207-223
  17. Jin, H.J. 1999. ermSF, a ribosomal RNA adenine N$_6$-methyltransferase gene from Streptomyces fradiae, confers MLS (macrolidelincosamide- streptogramin B) resistance to E. coli when it is expressed. Mol. Cells 9, 252-25
  18. Kovalic, D., R.B. Giannattasio, H.J. Jin, and B. Weisblum. 1994. 23S rRNA Domain V, a fragment that can be specifically methylated in vitro by the ErmSF (TlrA) methyltransferase. J. Bacteriol. 176, 6992-699
  19. Liu M. and S. Douthwaite. 2002. Methylation at nucleotide G745 or G748 in 23S rRNA distinguishes Gram-negative from Grampositive bacteria. Mol. Microbiol. 44, 195-204
  20. Rosteck Jr, R.R., P.A. Reynolds, and C.L. Hershberger. 1991. Homology between proteins controlling Streptomyces fradiaetylosin resistance and ATP-binding transport. Gene 102, 27-32.19
  21. Gandecha, A.R. and E. Cundliffe. 1996. Molecular analysis of tlrD, an MLS resistance determinant from tylosin producer, Streptomyces fradiae. Gene 180, 173-176
  22. Kamimiya, S. and B. Weisblum. 1988. Translation attenuation control of ermSF, an inducible resistance determinant encoding rRNA N-methyltransferase from Streptomyces fradiae. J. Bacteriol. 170, 1800-1811
  23. Birmingham, V.A., K.L. Cox, J.L. Larson, S.E. Fishman, C.L. Hershberger, and E.T. Seno. 1986. Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae. Mol. Gen. Genet. 204, 532-539
  24. Roberts, M.C., J. Sutcliffe, P. Courvalin, L.B. Jensen, J. Rood, and H. Seppala. 1999. Nomenclature for macrolide and macrolide-lincomycin- streptogramin B resistance determinants. Antimicrob. Agents Chemother. 43, 2823-2830