Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coli harboring APH-(3')-Ia were determined. The enzyme phosphorylates its substrates with kcat/Km values of 10(6)-10(8) M-1 s-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0-7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phosphorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water ("ATP hydrolase" activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on kcat/Km) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium-random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia.

Aminoglycoside 3'-phosphotransferases [APH(3')s] provide an important means for high-level resistance to neomycin-and kanamycin-type aminoglycoside antibiotics. A four-step purification which affords milligram quantities of homogeneous APH(3') type Ila [APH(3')-IIaJ is described. The kinetic parameters for the turnover of five substrates by the enzyme were determined, and the pH dependence and metal activation for catalysis were investigated. All five cysteines in the amino acid sequence of the enzyme exist in their reduced forms; hence, there are no disulfide bonds in the protein. Modification of the cysteine thiols by S-cyanylation showed essentially no effect on the enzymatic activity. A mutant enzyme derived from APH-3'-IIa, which possesses a conservative Glu-182-Asp point mutation and which provides diminished resistance to G418 (R. L. Yenofsky, M. Fine, and J. W. Pellow, Proc. Natl. Acad. Sci. USA 87:3435-3439, 1990), was also purified to homogeneity. Kinetic Among the enzymes that modify aminoglycoside antibiotics, aminoglycoside 3'-phosphotransferases [i.e., APH(3')s] are commonly found in resistant bacteria (4, 18, 27, 32). APH(3')s catalyze the transfer of the y-phosphoryl group of ATP to the 3'-hydroxyl group of kanamycins, neomycins, paromomycins, neamine, paromamine, ribostamycin, butirosin, and G418. Seven isozymes of APH(3') have been reported in the literature to date (for reviews, see references 4, 18, 28, and 31).In our effort toward a systematic structural and mechanistic analysis of the bacterial aminoglycoside phosphotransferases, we developed a practical purification for APH-3' type Ila [APH(3')-IIa] which affords several milligrams of homogeneous enzyme. Furthermore, we report here on the basic characterization of this enzyme. In addition, we report on the kinetic behavior of a mutant derivative of APH(3')-IIa with a conservative Glu-182-Asp substitution, which conferred a lower level of aminoglycoside resistance in vivo (33). MATERUILS AND METHODSMolecular mass markers and the Bio-Rad protein assay dye reagent concentrate were purchased from Bio-Rad. Kanamycin, neomycin, G418, amikacin, 5,5'-dithiobis(2-nitrobenzoic acid (DTNB), ATP, ADP, phosphoenolpyruvate (PEP), NADH, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES) were obtained from the Sigma Chemical Co. The cyanylating reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) was synthesized by the procedure described by Degani and Patchornik (9). Pyruvate kinase (PK) and lactate dehydroge-

No abstract

No abstract