The aminoglycoside antibiotic streptomycin has been important in the treatment of infection by a number of pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Over the years, many bacteria have developed resistance to streptomycin. One mechanism by which resistance can arise is acquisition of the gene for an enzyme that modifies the antibiotic, thereby rendering it inactive. Aminoglycoside phosphotransferase APH(6)-Id, which catalyzes the ATP-dependent phosphorylation of streptomycin, is such an enzyme. As part of an overall project to characterize APH(6)-Id in terms of its structural and functional properties, we have cloned the aph(6)-Id gene, and have expressed, purified and initially characterized the recombinant enzyme as a His-tagged fusion protein. The purified enzyme was assayed for activity using three methods: (1) a spectrophotometric method that utilizes a coupled assay; (2) an HPLC-based method that measures the consumption of streptomycin over time; and (3) a method that detects production of gamma-³²P-labeled streptomycin phosphate trapped by filtration through phosphocellulose paper. The first method seemed to indicate that the recombinant enzyme merely hydrolyzed ATP but did not produce streptomycin phosphate. The other two methods, however, showed that streptomycin was being consumed and streptomycin phosphate was being produced, proving that the enzyme is a true streptomycin phosphotransferase. Preliminary steady state kinetic analysis gave a Km (streptomycin) of 0.42 ± 0.13 mM and a Vmax of 1.5 ± 0.7 umol/min/mg. Experiments are now underway to determine more complete steady state kinetic constants for APH(6)-Id. Supported by NIH grants 2-G12-RR003048 and 1-SC3-GM083752-01