STRUCTURAL STUDIES ON AMINOGLYCOSIDE PHOSPHOTRANSFERASES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major factor in the emergence of antibiotic resistance is the existence of bacterial enzymes that chemically modify common antibiotics. One such family of anti-bacterials to which there is now almost universal resistance is the aminoglycosides (e.g. kanamycin, tobramycin and gentimicin). High level resistance to gentamicin in enterococci is mediated by a group of four phosphotransferases belonging to the APH(2?) sub-family of enzymes which phosphorylate at a specific hydroxyl group on the antibiotic. Structurally, the aminoglycoside phosphotransferase enzymes have a two-domain architecture resembling the catalytic subunit of the eukaryotic Ser/Thr and Tyr protein kinases. An understanding of how these enzymes bind and deactivate the aminoglycosides will provide valuable information for the design of specific inhibitors of these enzymes. We are studying all four of the APH(2?) phosphotransferases, APH(2?)-Ia, APH(2?)-Ic and APH(2?)-Id from Enterococcus faecium and APH(2?)-Ic from E. gallinarum. The three dimensional structures of the APH(2?)-Ib and APH(2?)-Ic enzymes have been determined. In addition, three wavelength MAD data has been collected from a SeMet APH(2?)-Ia crystal and the structure determination is underway. More recently diffraction quality crystals of APH(2?)-Id have been grown and some data collected.