Hydrolysis of ATP by Aminoglycoside 3′-Phosphotransferases

Abstract

Aminoglycoside 3-phosphotransferases (APH(3)s) are common bacterial resistance enzymes to aminoglycoside antibiotics. These enzymes transfer the ␥-phosphoryl group of ATP to the 3-hydroxyl of the antibiotics, whereby the biological activity of the drugs is lost. Pre-steady-state and steady-state kinetics with two of these enzymes from Gram-negative bacteria, APH(3)-Ia and APH(3)-IIa, were performed. It is demonstrated that these enzymes in both ternary and binary complexes facilitate an ATP hydrolase activity (ATPase), which is competitive with the transfer of phosphate to the antibiotics. Because these enzymes are expressed constitutively in resistant bacteria, the turnover of ATP is continuous during the lifetime of the organism both in the absence and the presence of aminoglycosides. Concentrations of the enzyme in vivo were determined, and it was estimated that in a single generation of bacterial growth there exists the potential that this activity would consume as much as severalfold of the total existing ATP. Studies with bacteria harboring the aph(3)-Ia gene revealed that bacteria are able to absorb the cost of this ATP turnover, as ATP is recycled. However, the cost burden of this adventitious activity manifests a selection pressure against maintenance of the plasmids that harbor the aph(3)-Ia gene, such that ϳ50% of the plasmid is lost in 1500 bacterial generations in the absence of antibiotics. The implication is that, in the absence of selection, bacteria harboring an enzyme that catalyzes the consumption of key metabolites could experience the loss of the plasmid that encodes for the given enzyme.

Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2′′-phosphotransferases

“…In all cases it is assumed that the -phosphate of the nucleotide is transferred to water. Similar residual levels of ATPase activity (k cat = 0.02 s À1 ) have previously been observed for members of the APH(3 0 ) subfamily, and it was demonstrated that wasteful GTP hydrolysis by these enzymes is evolutionarily disadvantageous for bacteria (Kim et al, 2006). The observation that the GTP complexes of APH(2 00 )-Ia (Caldwell et al, 2016;Smith et al, 2014) and APH(2 00 )-IIIa (Smith et al, 2012) have electron density consistent with GDP confirms that hydrolysis has occurred in the absence of substrate.…”

“…This phenomenon was first demonstrated for the APH(3 0 )-Ia and APH(3 0 )-IIa enzymes. They perform continuous basal-level ATP hydrolysis irrespective of whether an aminoglycoside substrate is present or not, which imposes a significant cost to the bacteria harboring the aph(3 0 ) gene (Kim et al, 2006). Subsequently, it was shown that two members of the APH(2 00 ) family, APH(2 00 )-Ia (Frase et al, 2012) and APH(2 00 )-IIIa (Badarau et al, 2008), also have a continuous intrinsic level of GTP hydrolysis in the absence of a substrate.…”

Embedded elements in the IncPβ plasmids R772 and R906 can be mobilized and can serve as a source of diverse and novel elements

“…They also imply that the maintenance of R772 in its clinical strain (Coetzee, 1978), and since then in laboratory strains, was due to aminoglycoside selection pressure. Indeed, the latter may be a strict requirement, since expression of aphAI imposes a cost burden on its bacterial host (Kim et al, 2006).…”

Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic, structural and kinetic study

“…It implies that ADP release, step 4 or 5′, is rate-limiting. We note that with three different APH(3′), a product release step is also rate limiting [27] , [46] , [47] .…”