Purification and characterization of aminoglycoside 3'-phosphotransferase type IIa and kinetic comparison with a new mutant enzyme

Abstract

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-

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase‐2″‐IVa

“…APH(2 00 )-IVa operates via a sequential mechanism in which the two substrates (NTP and aminoglycoside) bind in a random manner. All aminoglycoside-modifying enzymes studied to date (APH(3 0 )-Ia, APH(3 0 )-IIa, APH(2 00 )-Ia, APH(2 00 )-IIa, and APH(2 00 )-IIIa) operate via the sequential mechanism, 21,23,[26][27][28][29][30][31][32][33] and use a random mechanism for turnover chemistry, 21,23,31,33,34 with the exception of APH(3 0 )-IIIa, which has been shown to use a Theorell-Chance mechanism with compulsory ordered substrate binding and product release. 35 …”

“…34 Reaction mixtures containing 100 mM MES (pH 6.6), 10 mM MgCl 2 , 20 mM KCl, 4 mM phospho(enol)pyruvate, 200 lM b-nicotinamide adenine dinucleotide (reduced form), 20 U/mL pyruvate kinase, 25 U/mL lactate dehydrogenase, 0.1-2 mM ATP or GTP, the substrate analog (during inhibition experiments), and APH(2 00 )-IV (100-300 nM) were initiated by the addition of the aminoglycoside substrate, and the change in absorbance monitored at 340 nm.…”

Aminoglycoside 2″-Phosphotransferase Type IIIa from Enterococcus

“…Kinetic Studies-Enzyme activity was monitored (unless otherwise stated) by coupling the release of NDP (GDP, ADP, UDP, IDP, CDP, or TDP) from the APH(2Љ)-IIIa-catalyzed phosphorylation of the aminoglycosides to pyruvate kinase and lactate dehydrogenase, as previously described (5). In a typical experiment, 480 l of assay mixture (100 mM buffer, 100 mM NaCl, 10 mM MgCl 2 , 20 mM KCl, 0.2 mM NADH, 2 mM PEP, 12 units of pyruvate kinase, 27 units of lactate dehydrogenase) was mixed with NTP and APH(2Љ)-IIIa (23-230 nM); the reaction was started by adding the aminoglycoside.…”

“…This mechanism of resistance is particularly relevant for clinical enterococcal and staphylococcal isolates (2). Despite the widespread occurrence of aminoglycoside phosphotransferases in pathogenic bacteria, only a few of these enzymes have been characterized mechanistically (3)(4)(5)(6). The most prevalent aminoglycoside phosphotransferases are the APH(3Ј)s, which transfer a phosphate group to the 3Ј-hydroxyl of kanamycin A (7).…”

New aminoglycoside-modifying enzymes APH(3′)-VIII and APH(3′)-IX in Acinetobacter rudis and Acinetobacter gerneri

“…It has also been shown that APHs can phosphorylate several eukaryotic protein kinase substrates on serine residues. 14 In addition, APH enzymes are able to catalyze hydrolysis of ATP; 15,16 as these enzymes are produced constitutively and the turnover of ATP leads to a fitness cost. 17 Phosphorylation involves enzymatic transfer of the γ-phosphate of ATP to an hydroxyl group.…”