Spectinomycin kinase from Legionella pneumophila. Characterization of substrate specificity and identification of catalytically important residues

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Publication Date


Document Type



Adenosine; Adenosine Triphosphate; Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance; Kinetics; Legionella pneumophila; Legionnaires' Disease; Magnetic Resonance Spectroscopy; Mutagenesis, Site-Directed; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Sequence Alignment; Substrate Specificity


Biochemistry | Enzymes and Coenzymes | Medicinal-Pharmaceutical Chemistry | Therapeutics


The bacterium Legionella pneumophila is the responsible agent for Legionnaires' disease and has recently been shown to harbor a gene encoding a kinase that confers resistance to the aminoglycoside antibiotic spectinomycin (Suter, T. M., Viswanathan, V. K., and Cianciotto, N. P. (1997) Antimicrob. Agents Chemother. 41, 1385-1388). We report the overproduction, purification, and characterization of this spectinomycin kinase from an expressing system in Escherichia coli. The purified protein shows stringent substrate specificity for spectinomycin with Km = 21.5 microM and kcat = 24.2 s-1 and does not bind other aminoglycosides including kanamycin, amikacin, neomycin, butirosin, streptomycin, or apramycin. Purification of spectinomycin phosphate followed by characterization by mass spectrometry and 1H, 13C, and 31P NMR established the site of phosphorylation to be at the hydroxyl group at position 9. Thus this enzyme is designated APH(9)-Ia (where APH is aminoglycoside kinase). The enzyme was inactivated by the electrophilic ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, consistent with a nucleophilic residue such as Lys lining the nucleotide binding pocket. Site-directed mutagenesis of Lys-52 and Asp-212 to Ala confirmed that these residues were important for catalysis, with Lys-52 playing a potential role in ATP binding and Asp-212 in phosphoryl transfer. Thio and solvent 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 results establish that APH(9)-Ia is a highly specific antibiotic resistance kinase and provides the requisite mechanistic information for future structural studies.

DOI of Published Version



J Biol Chem. 1998 Jun 12;273(24):14788-95. doi:10.1074/jbc.273.24.14788

Journal/Book/Conference Title

The Journal of biological chemistry


At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.

Related Resources

Link to Article in PubMed