Aminoglycoside-modifying enzymes are responsible for high-level bacterial resistance to the aminoglycoside antiobiotics and compromise the effectiveness of these drugs for treating serious infections. It is likely that via mutational alteration the genes for these enzymes provide a reservoir from which bacterial resistance to newly designed aminoglycosides might emerge. This study proposes experiments aimed at characterizing the active site for an aminoglycoside 3'-phosphotransferase [APH(3')-II]. This enzyme normally confers bacterial resistance to kanamycin, but not to its semi-synthetic derivative, amikacin. This is true even though the enzyme modifies amikacin, albeit with much less catalytic efficiency than for kanamycin. Further kinetic studies of the purified APH(3')-II will be undertaken to identify potential inhibitors. Additionally, available sequence data has been examined and a number of conserved amino acids between several APH(3') enzymes have been identified. Modifying reagents specific for certain residues have been used to treat APH(3')-II to determine which of these are necessary for function. Thusfar histidine has been implicated in binding the AG, while tyrosine seems primarily to bind the ATP. The ATP-binding site will be further probed specifically using photoaffinity analogs of ATP. Together with site-directed mutagenesis of the cloned APH(3')-II gene, these approaches should provide us with insight as to the structure and function of this class of enzymes. Perhaps this will aid in determining the genetic potential of such enzymes to improve their recognition of new substrates, such as amikacin.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026579-02
Application #
3140360
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1990-05-01
Project End
1993-04-30
Budget Start
1991-05-01
Budget End
1992-04-30
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Louisville
Department
Type
Schools of Arts and Sciences
DUNS #
City
Louisville
State
KY
Country
United States
Zip Code
40292
Kocabiyik, S; Mullins, C; Breeding, C et al. (1992) Structure-function analyses for aminoglycoside 3'-phosphotransferase II (APH(3')-II). SAAS Bull Biochem Biotechnol 5:58-63
Kocabiyik, S; Perlin, M H (1992) Altered substrate specificity by substitutions at Tyr218 in bacterial aminoglycoside 3'-phosphotransferase-II. FEMS Microbiol Lett 72:199-202