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Pilot Project #1—
Structural and Functional Characterization
of Aminoglycoside 6-Phosphotransferases

W. Malcolm Byrnes, Ph.D.

Department of Biochemistry and Molecular Biology

The overall objective of this project is to provide detailed information about the structure and function of a group of aminoglycoside modifying enzymes, the aminoglycoside 6-phosphotransferases,that confer resistance to streptomycin or its close relatives. Streptomycin acts by binding to the A site of the 16S ribosomal RNA molecule; this blocks translocation. The 6-phosphotransferases inactivate the antibiotic by catalyzing the ATP-dependent phosphorylation of streptomycin at position 6 of the streptidine ring.

The focus is on two members of this group: the enzyme APH (6)-1a, whose gene is found within the streptomycin biosynthetic cluster of Streptomyces griseus, a common soil bacterium; and the enzyme APH(6)-ld, found on the broad host-range plasmid RSF1010 within human pathogenic bacterial isolates. The rationale for selecting these particular enzymes, APH(6)-la and –ld, is that, although similar in terms of the product they generate, they are different in terms of their source, i.e., non-pathogenic versus pathogenic bacteria, and their ecological role. It is expected that a comparison between them and with other aminoglycoside phosphotransferases will yield important information about what, if anything, mechanistically and structurally distinguishes the enzyme from nonpathogenic (S. griseus) and pathogenic (E. coli and others) bacteria.

The specific aims are to:

  1. obtain the genes for the two enzymes, express their gene products in Escherichia coli, and characterize them in terms of their regiospecificity using NMR and their detailed kinetic mechanisms applying steady-state kinetics;
  2. identify the key amino acid residues of the two enzymes that are involved in catalysis, ATP binding, streptomycin binding, and domain association; and
  3. determine the three dimensional structure of either APH(6)-Ia or -Id.

The detailed information gathered here about the structure and function of the streptomycin 6-phosphotransferases will extend our overall understanding of how aminoglycoside phosphotransferases bind and modify their substrates, which will aid in the design of general inhibitors of this class of resistance enzymes. Insights gained in this work also may give clues about how aminoglycoside resistance spreads within bacterial communities. This project utilizes resources of both the BNMR laboratory and the LMCB.

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