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 are 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, using ATP as a cosubstrate. 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 and APH(2?)-IIa from Enterococcus faecium, APH(2?)-IIIa from E. gallinarum, and APH(2?)-IVa from E. casseliflavus. The three dimensional structures of the binary gentamicin complex and a ternary AMPPCP-streptomycin complex of APH(2?)-IIa has been determined and recently published and the other crystallization conditions of APH(2?)-IIIa and APH(2?)-IVa have been reported. Both the APH(2?)-IIIa and APH(2?)-IVa structures has been solved by molecular replacement using APH(2?)-IIa as the search model. In addition, three wavelength MAD data has been collected from a SeMet APH(2?)-Ia crystal and the structure has been solved and partially refined. Additional structural studies on binary and ternary complexes of these enzymes are underway.