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

Abstract

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2 00 -IVa (APH(2 00 )-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2 00 )-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k cat /K m ) of 1.5 3 10 3 to 1.2 3 10 6 (M 21 s 21 ). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2 00 )-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

“…We and others have shown that APH(2Љ)-IIa and -IVa have comparable relative affinities for both ATP and GTP, whereas the two others preferentially bind GTP, an indication that GTP and not ATP is the physiological cosubstrate for these enzymes (9,10). Contrary to what has been reported for the APH(2Љ)-Ia domain of the bifunctional enzyme, our kinetic characterization of APH(2Љ)-IIa, -IIIa, and -IVa demonstrated that 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates, but inhibitors of these enzymes (11)(12)(13). In light of the substantial differences in the reported substrate specificities of APH(2Љ)-Ia and the remaining members of the APH(2Љ) subfamily of aminoglycoside kinases and the discovery that GTP, and not ATP, is the preferred cosubstrate for APH(2Љ)-Ia, we sought to reevaluate the nucleotide and aminoglycoside substrate profiles of the clinically important AAC(6Ј)-Ie/APH(2Љ)-Ia enzyme.…”

“…Recent kinetic and x-ray crystallographic studies of three APH(2Љ) phosphotransferases, APH(2Љ)-IIa, -IIIa, and -IVa, revealed that these enzymes possess structural templates for the binding of both ATP and GTP (13,25,26). The availability of both structural templates accounts for the ability of APH(2Љ)-IIa and -IVa to use both ATP and GTP as cosubstrates.…”

Novel Aminoglycoside 2″-Phosphotransferase Identified in a Gram-Negative Pathogen

“…Regardless, the 4,5-disubstituted and atypical aminoglycosides are at most very poor substrates for the APH(2Љ)-If and APH(2Љ)-Ia phosphotransferases, and this finding is consistent with the MIC data, which show no resistance to these antibiotics (Table 1). These data are also in good agreement with the substrate profiles of three other APH(2Љ) enzymes, none of which showed the ability to efficiently phosphorylate 4,5-disubstituted or atypical aminoglycosides (22,24,25). Next, we tested neomycin B, paromomycin, lividomycin A, and neamine as inhibitors, and overall, 4,5-disubstituted aminoglycosides are better inhibitors of APH(2Љ)-If than the APH(2Љ)-Ia domain of the bifunctional enzyme (Table 3).…”

“…Members of this enzyme family, APH(2Љ)-Ia, -IIa, -IIIa, and -IVa are bisubstrate enzymes that phosphorylate aminoglycoside antibiotics using GTP and/or ATP as the phosphate donor (23). The recently determined X-ray crystal structures of three of these enzymes, APH(2Љ)-IIa, -IIIa, and -IVa, have shed light on substrate recognition and NTP selectivity by these aminoglycoside kinases (24,30,31). In our quest for novel APH(2Љ) phosphotransferases, we analyzed the amino acid sequences of enzymes deposited in the database of the National Center for Biotechnology Information.…”

“…Four distantly related APH(2Љ) subfamilies, sharing 28 to 32% amino acid sequence identity, have been identified in clinical enterococcal isolates (21). Recently, a novel nomenclature for these enzymes has been proposed based on their detailed kinetic characterization (22)(23)(24)(25). The enzymes were renamed according to the subfamily into which they belong: thus, APH(2Љ)-Ib, -Ic, and -Id are now referred to as APH(2Љ)-IIa, -IIIa, and -IVa, respectively.…”