Nucleotide Selectivity of Antibiotic Kinases

Abstract

Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3)-IIIa, APH(2؆)-Ib, and MPH(2)-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3)-IIIa exclusively uses ATP, MPH(2)-I exclusively uses GTP, and APH(2؆)-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.Antibiotic modification is a major mechanism of resistance that impacts the efficacy of numerous antimicrobial drug classes. The enzymes that catalyze these group transfer mechanisms have evolved from precursor genes that encode proteins used to accomplish numerous metabolic tasks no doubt unrelated to drug resistance. Borrowing nomenclature from the oncogene field, we term such elements protoresistance genes. Biochemical and structural evidence has shown that protoresistance genes can be found in a number of key metabolic pathways, including cell wall biosynthesis and signal transduction (23). With respect to group transfer antibiotic resistance enzymes, these protoresistance genes encode protein and other small-molecule kinases, acetyltransferases, adenylyltransferases, ADP-ribosyltransferases, and glycosyltransferases. The addition of phosphoryl, acyl, adenyl, ribosyl, and glycosyl groups to the antibiotic scaffold alters the interaction with the cellular target to such a degree that the resistance phenotype results (6).The unifying biochemical logic of group transfer in antibiotic resistance is the co-opting of the normal cellular function of the protoresistance element by natural selection to include the modification of antibiotic molecules. Because group transfer enzymes require a second substrate (e.g., ATP, acetyl coenzyme A, or thymidine diphosphate glucose), these should retain the original specificity of the protoresistance enzyme since natural selection would not normally be expected to act on this site during resistance gene evolution.Antibiotic kinases represent a large superfamily of enzymes that covalently modify antibiotic...

Resistance to Macrolide Antibiotics in Public Health Pathogens

“…Macrolide 2 0 -phosphotransferases, commonly found on mobile genetic elements, are inducible (e.g., mph(A)) or constitutively expressed (e.g., mph(B)) intracellular enzymes capable of transferring the g-phosphate of nucleotide triphosphate to the 2 0 -OH group of 14-, 15-, and 16-membered-ring macrolide antibiotics, thereby disrupting the macrolide's key interaction with A2058. Although early studies showed Mph enzymes could use ATP, more recent work with Mph(A) has shown a preference for GTP under physiologically relevant in vitro assay conditions (Shakya and Wright 2010). Expression of mph(A) is induced by erythromycin, and, recently, the structure of the MphR(A) repressor protein, a negative regulator of mph(A) expression, has been solved uncomplexed and complexed with erythromycin to 2.00 Å and 1.76 Å resolutions, respectively (Zheng et al 2009).…”

“…Macrolide phosphotransferases are macrolideinactivating enzymes widespread in Gram-negative and Gram-positive bacteria (Sutcliffe and Leclercq 2002;Roberts 2008) that, by in silico analysis, are in the same family as aminoglycoside and protein kinases (Shakya and Wright 2010). The first reported purifications of macrolide-2 0 -phosphotransferases were from macrolide-resistant E. coli, and this mechanism was soon shown to be prevalent in E. coli clinical isolates in Japan (O'Hara et al 1989;Kono et al 1992;Taniguchi et al 2004).…”

“…In addition, macrolides can be inactivated by esterases or phosphotransferases (in public health pathogens and macrolide producers) or by glycosyltransferases (decribed in many strains of Streptomyces producing polyketides or polyether antibiotics; Micromonospora purpurea; Nocardia asteroides), deacylases (N. asteroides), or formyl reductases (N. asteroides, Nocardia brasiliensis, Nocardia otitidiscaviarum) (Sutcliffe and Leclercq 2002;Roberts 2008;Shakya and Wright 2010;Morar et al 2012). Many strains carry more than one macrolide resistance mechanism, sometimes on the same mobile element.…”

Aminoglycoside 2′′-Phosphotransferase IIIa (APH(2′′)-IIIa) Prefers GTP over ATP

“…Our recent work on the APHs from the aminoglycoside-2ЈЈ-phosphotransferase [APH(2ЈЈ)] subfamily (25) have shown that not only are these enzymes capable of using GTP as the phosphate source but that two members of this family (APH(2ЈЈ)-Ia and APH(2ЈЈ)-IIIa) utilize GTP exclusively in vivo. The ability of the APH(2ЈЈ) enzymes to use GTP as a cofactor is unexpected, as APHs were also considered to be exclusively ATP-dependent (20,26). This raises a number of questions related to nucleotide binding and catalysis in the aminoglycoside kinases and in the protein kinases in general.…”

Structure and Function of APH(4)-Ia, a Hygromycin B Resistance Enzyme

“…In contrast, APH(4)-Ia demonstrated the ability to utilize both ATP and GTP as a phosphate donor with ϳ5-fold preference toward ATP based upon the k cat /K m values of 5.42 ϫ 10 4 and 1.04 ϫ 10 4 , respectively. Similar nucleotide promiscuity has been previously observed for the APH(2Љ) class of enzymes (31,32). In eukaryotic protein kinases, the capacity to utilize GTP is rare, and this difference could be exploited in the development of specific inhibitors.…”