Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 May;174(10):3196–3203. doi: 10.1128/jb.174.10.3196-3203.1992

Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 6'-N-acetyltransferase Ib and IIa proteins.

P N Rather 1, H Munayyer 1, P A Mann 1, R S Hare 1, G H Miller 1, K J Shaw 1
PMCID: PMC205986  PMID: 1577689

Abstract

The aminoglycoside 6'-N-acetyltransferase [AAC(6')-I] and AAC(6')-II enzymes represent a class of bacterial proteins capable of acetylating tobramycin, netilmicin, and 2'-N-ethylnetilmicin. However, an important difference exists in their abilities to modify amikacin and gentamicin. The AAC(6')-I enzymes are capable of modifying amikacin. In contrast, the AAC(6')-II enzymes are capable of modifying gentamicin. Nucleotide sequence comparison of the aac(6')-Ib gene and the aac(6')-IIa gene showed 74% sequence identity (K. J. Shaw, C. A. Cramer, M. Rizzo, R. Mierzwa, K. Gewain, G. H. Miller, and R. S. Hare, Antimicrob. Agents Chemother. 33:2052-2062, 1989). Comparison of the deduced protein sequences showed 76% identity and 82% amino acid similarity. A genetic analysis of these two proteins was initiated to determine which amino acids were responsible for the differences in specificity. Results of domain exchanges, which created hybrid AAC(6') proteins, indicated that amino acids in the carboxy half of the proteins were largely responsible for determining specificity. Mutations shifting the specificity of the AAC(6')-Ib protein to that of the AAC(6')-IIa protein (i.e., gentamicin resistance and amikacin sensitivity) have been isolated. DNA sequence analysis of four independent isolates revealed base changes causing the same amino acid substitution, a leucine to serine, at position 119. Interestingly, this serine occurs naturally at the same position in the AAC(6')-IIa protein. Oligonucleotide-directed mutagenesis was used to construct the corresponding amino acid change, a serine to leucine, in the AAC(6')-IIa protein. This change resulted in the conversion of the AAC(6')-IIa substrate specificity to that of AAC(6')-Ib. Analysis of additional amino acid substitutions within this region of AAC(6')-Ib support the model that we have identified an aminoglycoside binding domain of these proteins.

Full text

PDF
3196

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benveniste R., Davies J. Mechanisms of antibiotic resistance in bacteria. Annu Rev Biochem. 1973;42:471–506. doi: 10.1146/annurev.bi.42.070173.002351. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Champion H. M., Bennett P. M., Lewis D. A., Reeves D. S. Cloning and characterization of an AAC(6') gene from Serratia marcescens. J Antimicrob Chemother. 1988 Nov;22(5):587–596. doi: 10.1093/jac/22.5.587. [DOI] [PubMed] [Google Scholar]
  4. Courvalin P., Carlier C. Resistance towards aminoglycoside-aminocyclitol antibiotics in bacteria. J Antimicrob Chemother. 1981 Jul;8 (Suppl A):57–69. doi: 10.1093/jac/8.suppl_a.57. [DOI] [PubMed] [Google Scholar]
  5. Davies J. General mechanisms of antimicrobial resistance. Rev Infect Dis. 1979 Jan-Feb;1(1):23–29. doi: 10.1093/clinids/1.1.23. [DOI] [PubMed] [Google Scholar]
  6. Davies J., Smith D. I. Plasmid-determined resistance to antimicrobial agents. Annu Rev Microbiol. 1978;32:469–518. doi: 10.1146/annurev.mi.32.100178.002345. [DOI] [PubMed] [Google Scholar]
  7. Gros P., Dhir R., Croop J., Talbot F. A single amino acid substitution strongly modulates the activity and substrate specificity of the mouse mdr1 and mdr3 drug efflux pumps. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7289–7293. doi: 10.1073/pnas.88.16.7289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haas M. J., Dowding J. E. Aminoglycoside-modifying enzymes. Methods Enzymol. 1975;43:611–628. doi: 10.1016/0076-6879(75)43124-x. [DOI] [PubMed] [Google Scholar]
  9. Haas M., Biddlecome S., Davies J., Luce C. E., Daniels P. J. Enzymatic modification of aminoglycoside antibiotics: a new 6'-N-acetylating enzyme from a Pseudomonas aeruginosa isolate. Antimicrob Agents Chemother. 1976 Jun;9(6):945–950. doi: 10.1128/aac.9.6.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Juvonen R. O., Iwasaki M., Negishi M. Structural function of residue-209 in coumarin 7-hydroxylase (P450coh). Enzyme-kinetic studies and site-directed mutagenesis. J Biol Chem. 1991 Sep 5;266(25):16431–16435. [PubMed] [Google Scholar]
  12. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Leslie A. G. Refined crystal structure of type III chloramphenicol acetyltransferase at 1.75 A resolution. J Mol Biol. 1990 May 5;213(1):167–186. doi: 10.1016/S0022-2836(05)80129-9. [DOI] [PubMed] [Google Scholar]
  14. Lovering A. M., White L. O., Reeves D. S. AAC(1): a new aminoglycoside-acetylating enzyme modifying the Cl aminogroup of apramycin. J Antimicrob Chemother. 1987 Dec;20(6):803–813. doi: 10.1093/jac/20.6.803. [DOI] [PubMed] [Google Scholar]
  15. McClary J. A., Witney F., Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. Biotechniques. 1989 Mar;7(3):282–289. [PubMed] [Google Scholar]
  16. Radika K., Northrop D. B. Substrate specificities and structure-activity relationships for acylation of antibiotics catalyzed by kanamycin acetyltransferase. Biochemistry. 1984 Oct 23;23(22):5118–5122. doi: 10.1021/bi00317a006. [DOI] [PubMed] [Google Scholar]
  17. Radika K., Northrop D. B. The kinetic mechanism of kanamycin acetyltransferase derived from the use of alternative antibiotics and coenzymes. J Biol Chem. 1984 Oct 25;259(20):12543–12546. [PubMed] [Google Scholar]
  18. Shaw K. J., Cramer C. A., Rizzo M., Mierzwa R., Gewain K., Miller G. H., Hare R. S. Isolation, characterization, and DNA sequence analysis of an AAC(6')-II gene from Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1989 Dec;33(12):2052–2062. doi: 10.1128/aac.33.12.2052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sowek J. A., Singer S. B., Ohringer S., Malley M. F., Dougherty T. J., Gougoutas J. Z., Bush K. Substitution of lysine at position 104 or 240 of TEM-1pTZ18R beta-lactamase enhances the effect of serine-164 substitution on hydrolysis or affinity for cephalosporins and the monobactam aztreonam. Biochemistry. 1991 Apr 2;30(13):3179–3188. doi: 10.1021/bi00227a004. [DOI] [PubMed] [Google Scholar]
  20. Tenover F. C., Filpula D., Phillips K. L., Plorde J. J. Cloning and sequencing of a gene encoding an aminoglycoside 6'-N-acetyltransferase from an R factor of Citrobacter diversus. J Bacteriol. 1988 Jan;170(1):471–473. doi: 10.1128/jb.170.1.471-473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Terán F. J., Suárez J. E., Mendoza M. C. Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile. Antimicrob Agents Chemother. 1991 Apr;35(4):714–719. doi: 10.1128/aac.35.4.714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tran van Nhieu G., Collatz E. Primary structure of an aminoglycoside 6'-N-acetyltransferase AAC(6')-4, fused in vivo with the signal peptide of the Tn3-encoded beta-lactamase. J Bacteriol. 1987 Dec;169(12):5708–5714. doi: 10.1128/jb.169.12.5708-5714.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Vermersch P. S., Lemon D. D., Tesmer J. J., Quiocho F. A. Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity. Biochemistry. 1991 Jul 16;30(28):6861–6866. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES