TOWARDS UNRAVELLING THE STRUCTURE OF AN ANTIBIOTICS-INACTIVATING ENZYME - KANAMYCIN KINASE. Wai-Ching Hon, Gerard D Wright, Daniel S-C Yang & Albert M Berghuis, Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada. L8N 3Z5

The enterococcal kanamycin kinase (3', 5"-aminoglycoside phosphotransferase type IIIa (APH(3')) belongs to a class of enzymes that inactivate the aminoglycoside-aminocyclitol type of antibiotics. It has a promiscuous substrate spectrum and can regiospecifically phosphorylate the 3' and/or 5" hydroxyl group of eight aminoglycosides, some of which are clinically important. High resolution structural models of the enzyme is imperative to complement biochemical analyses in fully understanding its kinetic and chemical mechanisms. The eventual goal of this work is the development of more effective antibiotics or inhibitors to this enzyme.

We have overexpressed and purified the protein from E. coli. Crystals that are complexed with the enzyme's various cofactors, substrates and their analogs have been obtained. These crystals diffract on average to 2.7 Å. A full dataset of an ATP-cocrystal, which diffracted beyond 2.2 Å, has been collected at the X12C beamline at NSLS. Results of our work on the structure solution using the isomorphous replacement method will be presented.