Broad Spectrum Aminoglycoside Phosphotransferase Type III from Enterococcus: Overexpression, Purification, and Substrate Specificity

Abstract

The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibiotics. We report the characterization of an enterococcal enzyme, APH(3')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP-dependent O-phosphorylation. Overproduction of APH(3')-IIIa has permitted the isolation of 30-40 mg of pure protein/(L of cell culture). Purified APH(3')-IIIa is a mixture of monomer and dimer which is slowly converted to dimer only over time. Dimer could be dissociated into monomer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show Km values in the low micromolar range for good substrates such as kanamycin and neomycin, and kcat values of 1-4 s-1. All aminoglycosides show substrate inhibition except amikacin and kanamycin B. Determination of minimum inhibitory concentrations indicates a positive correlation between antibiotic activity and kcat/Km, but not with Km or kcat. NMR analysis of phosphorylated kanamycin A has directly demonstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of inhibitors of APH(3')-IIIa.

Structural Basis for Dual Nucleotide Selectivity of Aminoglycoside 2″-Phosphotransferase IVa Provides Insight on Determinants of Nucleotide Specificity of Aminoglycoside Kinases

“…Kinetic Assay-A continuous spectrophotometric assay that couples aminoglycoside phosphorylation with lactate production was used to measure the consumption of ATP or GTP as a function of NADH oxidation (19). Data were collected at 37°C with a SpectraMax 190 absorbance microplate reader, where each assay was performed with a total volume of 200 l, containing 50 mM Tris-HCl at pH 7.5, 40 mM potassium chloride, 10 mM magnesium chloride, 3.0 mM phosphoenolpyruvate, 3.0 mM tobramycin, 360 mM ␤-nicotinamide adenine dinucleotide (reduced), 20 units/ml pyruvate kinase, and 25 units/ml lactate dehydrogenase.…”

The COOH Terminus of Aminoglycoside Phosphotransferase (3′)-IIIa Is Critical for Antibiotic Recognition and Resistance

“…The assay is described in greater detail elsewhere (3). When the kinetic parameters of the D261A were determined for neomycin we found that the K m was near the detection limit for the assay described above.…”

“…The forward PCR primer has been described previously (3). The D261A, E262A, F264A, and D261A/⌬Phe 264 double mutant were similarly generated using the mutagenic oligonucleotides, 5Ј-CCCGAAGC-TTCTAAAACAATTCAGCCAGTAAAATATAATA-3Ј, 5Ј-CCCGAAGCT-TCTAAAACAATGCATCCAGTAAAAT-3Ј, 5Ј-CGAAGCTTGGATCCCT-AAGCCAATTCATCCA-3Ј and 5Ј-CCCGAAGCTTCTACAATTCAGCC-AGTAAAATATAATATTTTA-3Ј, respectively, as the reverse primers.…”

“…In this work, a Q-Sepharose column (Amersham Pharmacia Biotech) (2.5 ϫ 10 cm) was used in place of the MacroPrep Q column described (3). Purification of certain mutant APH(3Ј)-IIIa proteins required modifications of the purification protocol.…”

“…This enzyme has a broad aminoglycoside substrate specificity with the demonstrated capacity to phosphorylate aminoglycosides at position 3Ј, and for aminoglycosides with a pentose linked to position 6 of the 2-deoxyaminocyclitol ring, position 5Љ (see Fig. 1 for representative structures and ring numbering) (3,4). In addition to naturally occurring aminoglycosides (and their semi-synthetic derivatives), APH(3Ј)-IIIa has been shown to phosphorylate a series of de-aminated and deoxyaminoglycoside derivatives with higher tolerance than the related APH(3Ј)-Ia and APH(3Ј)-IIa which are found in Gram-negative bacteria (5).…”