SPECTINOMYCIN KINASE FROM LEGIONELLA-PNEUMOPHILA - CHARACTERIZATION OF SUBSTRATE-SPECIFICITY AND IDENTIFICATION OF CATALYTICALLY IMPORTANT RESIDUES

The bacterium Legionella pneumophila is the responsible agent for Legi onnaires' disease and has recently been shown to harbor a gene encodin g a kinase that confers resistance to the aminoglycoside antibiotic sp ectinomycin (Suter, T. M., Viswanathan, V. H., and Cianciotto, N. P. ( 1997) Antimicrob. Agents Chemother. 41, 1385-1388). We report the over production, purification, and characterization of this spectinomycin k inase from an expressing system in Escherichia coli. The purified prot ein shows stringent substrate specificity for spectinomycin with K-m = 21.5 mu m and k(cat) = 24.2 s(-1) and does not bind other aminoglycos ides including kanamycin, amikacin, neomycin, butirosin, streptomycin, or apramycin. Purification of spectinomycin phosphate followed by cha racterization by mass spectrometry and H-1, C-13, and P-31 NMR establi shed the site of phosphorylation to be at the hydroxyl group at positi on 9. Thus this enzyme is designated APH(S)-Ia (where APH is aminoglyc oside kinase). The enzyme was inactivated by the electrophilic ATP ana logue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, consistent with a nucle ophilic residue such as Lys lining the nucleotide binding pocket. Site -directed mutagenesis of Lys-52 and Asp-212 to Ala confirmed that thes e residues were important for catalysis, with Lys-52 playing a potenti al role in ATP binding and Asp-212 in phosphoryl transfer. Thio and so lvent isotope effect experiments in the presence of either Mg2+ or Mn2 + were consistent with a kinetic mechanism in which phosphate transfer does not contribute significantly to the rate-limiting step. These re sults establish that APII(9)-Ia is a highly specific antibiotic resist ance kinase and provides the requisite mechanistic information for fut ure structural studies.