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1994 Fiscal Year Final Research Report Summary

Studies on the Molecular Structure and Function of Tetracycline Efflux Protein

Research Project

Project/Area Number 05454619
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Structural biochemistry
Research InstitutionCHIBA UNIVERSITY

Principal Investigator

YAMAGUCHI Akihito  Chiba University Faculty of Pharm.Sci.Associate Professor, 薬学部, 助教授 (60114336)

Project Period (FY) 1993 – 1994
Keywordsdrug efflux / drug resistance / tetracycline / site-directed mutagenesis / antiporter
Research Abstract

Since 1990, there have been a lot of drug efflux proteins found in bacteria. So, the molecular biological studies on tetracycline efflux protein (Tet) , which is now a unique protein of which molecular mechanisms are studied, becomes more important as a paradigm of such drug efflux proteins. In this study, we revealed the molecular mechanism of Tet mainly by gene engineering methods.
There is a well-known sequence motif widely conserved in secondary membrane transporters such as mammalian glucose transporters, bacterial sugar/H^+ symporters and bacterial drug exporters, which is depicted as GXXSDRXGRR.However, the precise role of this motif in the transporter function is still unknown. We first studied the role of this motif in this study by using site-directed mutagenesis technique. As a result, the 5th Asp66 and 9th Arg70 are essential for the function. As to Arg70, Lys70 mutant retained about 30% the wild type transport activity, whereas the mutants to a neutral or acidic residues lost the transport activity except for Cys70 mutant. Cys70 mutant retained the activity comparable to Lys70 mutant. The unexpectedly high activity of Cys70 mutant is due to the mercaptide formation with a divalent cation which acts as a cationic side chain.
Among 8 Cys mutants of this motif, only Cys65 and Cys70 mutants were inactivated by NEM.The binding of NEM to these mutants was stimulated by addition of tetracycline, indicating that the motif, which is located on the cytoplasmic surface, is exposed to the medium by the substrate. In contrast, NEM binding to Cys97, which is located on the periplasmic surface, was inhibited by tetracycline, indicating that the periplasmic residue is hidden by a substrate-induced conformational change. These observations confirm our model for tetracycline/H^+ antiport, in which tetracycline makes Tet protein an inside-open/outside-closed conformation.

  • Research Products

    (17 results)

All Other

All Publications (17 results)

  • [Publications] A.Yamaguchi: "Metal-tetracycline/H^+ antiporter of Escherichia coli encoded by transposon Tn10: The stru-.." J.Biol.Chem.268. 6496-6504 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] A.Yamaguchi: "Roles of the conserved quartets of residues located in the N- and C-terminal halves of the..." Biochemistry. 32. 5698-5704 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] A.Yamaguchi: "Hot spot for sulfhydryl inactivation of Cys mutants in the widely conserved sequence motif.." J.Biochem.115. 958-964 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Someya: "Site-specificity of the second-site suppressor mutation of the Asp285-Asn mutant of..." Biochemistry. 34. 7-12 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Someya: "A novel glycylcycline,9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline.is..." Antimicrob.Agents Chemother.39. 247-249 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Kimura: "Substrate-induced acceleration of N-ethylmaleimide binding to the Cys-65 mutant..." FEBS Lett.in press.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 橋本一,井上松久編: "病原菌の薬剤耐性:機構の解明とその対策" 学会出版センター, 200 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kimura, T., Inagaki, Y., Sawai, T., and Yamaguchi, A.: "Substrate-induced acceleration of N-ethylmaleimide binding to the Cys-65 mutant of the transposon Tn10-encoded metal-tetracycline/H^+ antiporter depends on the interaction of Asp-66 with the substrate." FEBS Lett. (in press). (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Someya, Y., Niwa, A., Sawai, T.and Yamaguchi, A: "Site-specificity of the second-site-suppressor mutation of the Asp285->Asn mutant of Metal-tetracycline/H^+ antiporter of Escherichia coli and the effects of amino acid substitutions at the first and second sites." Biochemistry. 34. 7-12 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Someya, Y., Yamaguchi, A., and Sawai, T.: "A novel glycylcycline, 9- (N,N-dimethylglycylamido) -6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H^+ antiporter.Antimicrob." Agents Chemother.39. 247-249 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamaguchi, A., Kimura, T., and Sawai, T.: "Hot spot for sulfhydryl inactivation of Cys mutants in the widely conserved sequence motifs of the meta-tetracycline/H^+ antiporter of Escherichia coli" J.Biochem.115. 958-964 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sato, K., Sato, M.H., Yamaguchi, A., and Yoshida, M.: "Tetracycline/H^+ antiporter was degraded rapidly in Escherichia coli cells when truncated at transmembrane helix and this degradation was protected by overproduced GroEL/ES." Biochem.Biophys.Res.Commun.202. 258-264 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamaguchi, A., O'yauchi, R., Someya, Y., Akasaka, T., and Sawai, T.: "Second-Site Mutation of Ala-220 to Glu or Asp Suppresses the Mutation of Asp-285 to Asn in the Transposon Tn10-Encoded Metal-Tetracycline/H^+ Antiporter of Escherichia coli" J.Biol.Chem.268. 26990-26995 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamaguchi, A., Kimura, T., Someya, Y., and Sawai, T.: "Metal-Tetracycline/H^+ Antiporter of Escherichia coli Encoded by Transposon Tn10 : The Structural Resemblance and Functional Difference in the Role of the Duplicated Sequence Motif between Hydrophobic Segments 2 and 3, and Segments 8 and 9." J.Biol.Chem.268. 6496-6504 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamaguchi, A., Akasaka, T., Kimura, T., Sakai, T., Adachi, Y., and Sawai, T.: "Roles of the Conserved Quartets of Residues Located in the N-and C-Terminal Halves of the Transposon Tn10-Encoded Metal-Tetracycline/H^+ Antiporter of Escherichia coli." Biochemistry. 32. 5698-5704 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamaguchi, A., Someya, Y., and Sawai, T.: "The in vivo Assembly and Function of the N-and C-Terminal Halves of the Tn10-Encoded TetA Protein in Escherichia coli" FEBS Letters. 324. 131-135 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamaguchi, A., Kimura, T., and Sawai, T.: "Effects of Sulfhydryl Reagents on the Cys65 Mutant of the Transposon Tn10-Encoded Metal-Tetracycline/H^+ Antiporter of Escherichia coli." FEBS Letters. 322. 201-204 (1993)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1996-04-15  

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