Bulky “Gatekeeper” Residue Changes the Cosubstrate Specificity of Aminoglycoside 2″-Phosphotransferase IIa

Abstract

A minoglycoside antibiotics are broad-spectrum compounds used for treatment of serious infections caused by both Grampositive and Gram-negative bacterial pathogens (1). The major mechanism of aminoglycoside resistance in Gram-positive and Gram-negative bacteria is the production of aminoglycosidemodifying enzymes, aminoglycoside phosphotransferases (APHs; also known as aminoglycoside kinases), aminoglycoside acetyltransferases, and aminoglycoside nucleotidyltransferases. These enzymes modify hydroxyl or amino groups on the antibiotics that are important for their binding to the target, the 30S subunit of the bacterial ribosome, thus significantly compromising their antimicrobial activity (2). It has been demonstrated that the APHs are capable of phosphorylating the 4-, 6-, 9-, 3=-, 2Љ-, 3Љ-, and 7Љ-hydroxyl groups of various aminoglycosides (3), and until recently, it was widely accepted that ATP is the major source of phosphate for these enzymes. Comprehensive studies of the aminoglycoside 2Љ-phosphotransferases [APH(2Љ)], enzymes which are widely distributed in Gram-positive staphylococcal (4) and enterococcal (5-7) isolates, have demonstrated that they may utilize GTP as a cosubstrate (8). Based on this knowledge, a new nomenclature for the APH(2Љ) enzymes was proposed, which reclassified the APH(2Љ)-Ia, -Ib, -Ic, and -Id enzymes as APH(2Љ)-Ia, -IIa, -IIIa, and -IVa enzymes, respectively (8). Both APH(2Љ)-Ia and APH(2Љ)-IIIa utilize exclusively GTP as a cofactor, while APH(2Љ)-IIa and APH(2Љ)-IVa can utilize both ATP and GTP (8,9). At present, it is not known whether the ability to utilize GTP for phosphorylation of aminoglycoside antibiotics is limited to the aminoglycoside 2Љ-phosphotransferases or if the phenomenon is more widespread. It is also unclear whether the ability to utilize both ATP and GTP or one of the nucleoside triphosphates (NTPs) in preference to another provides any advantage to bacteria. The concentrations of both ATP and GTP in bacterial cells are maintained in a very high, millimolar range (10, 11), implying that both NTPs are constantly available.X-ray crystallographic studies were crucial for understanding the structural requirements for the NTP profiles of the APH(2Љ) enzymes, by revealing that these kinases possess overlapping but distinct structural templates for ATP and GTP binding (12,13).Of these two templates, the ATP-binding site is located deeper in the NTP-binding pocket of the enzymes. It was further demonstrated that the inability of APH(2Љ)-IIIa [and presumably APH(2Љ)-Ia] to utilize ATP as a cosubstrate stems from the obstruction of its ATP-binding template by a bulky "gatekeeper" tyrosine residue, Tyr92 (14). To better understand the structural requirements for NTP specificity in the APH(2Љ) kinases, we attempted to convert APH(2Љ)-IIa and APH(2Љ)-IVa (both are capable of utilizing ATP and GTP as cosubstrates) into exclusively GTP-utilizing kinases by blocking their ATP-binding templates. MATERIALS AND METHODSMutagenesis. The M85Y and F95Y substitutions were introduced ...

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues

“…In exclusive GTP-specific isoforms, Tyr blocks entry of ATP; thus, adding the Tyr gatekeeper residue to ATP-specific isoforms resulted in a high GTP affinity. 29 Computational analysis of DNA polymerase μ with its cognate and noncognate ligands has suggested the role of gatekeeper residues in tightening the nucleotide binding pocket, which in turn alters the electrostatic potential, in addition to active site distortion by crucial residues. 30 In β-lactamase (BlaC), Ile 105 acted as a gatekeeper residue controlling access of the substrate to active site.…”

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia

“…This indicates that removal of the phenolate side chain gives access to the adenine-specificity template and the mutant is able to bind ATP efficiently. The recent kinetic analysis of the M85Y mutant of APH(2 00 )-IIa had the opposite effect, switching the enzyme to a GTPexclusive variant with a more than 300-fold increase in the K m for ATP and a dramatic decrease in catalytic efficiency by three orders of magnitude (Bhattacharya et al, 2013). Here, we observe an interesting phenomenon with the Y100F mutant of APH(2 00 )-Ia, the effect of which is not as clear as the aforementioned mutations, yet which overall produces a mutant enzyme capable of conferring resistance to the aminoglycosides.…”

“…By contrast, in APH(2 00 )-IVa the corresponding secondary hydrophobic pocket residues [Val78 and Ala93; APH(2 00 )-IVa residue numbering] are less bulky, and this opens the secondary pocket significantly in this enzyme such that the phenylalanine gatekeeper can be readily accommodated. Structural analysis of the F95Y mutant of APH(2 00 )-IVa showed that the bulkier tyrosine could also be accommodated in the larger secondary pocket (Shi & Berghuis, 2012), and two independent kinetic analyses of F95Y mutants of APH(2 00 )-IVa demonstrate the ability of this mutant to utilize both GTP and ATP as cosubstrates (Shi & Berghuis, 2012;Bhattacharya et al, 2013).…”

“…In the case of the GTP-exclusive enzymes APH(2 00 )-Ia and APH(2 00 )-IIIa this residue is a tyrosine, whereas in APH(2 00 )-IIa and APH(2 00 )-IVa, which have an almost equal preference for ATP and GTP, this gatekeeper residue is a methionine and a phenylalanine, respectively. It was suggested that the tyrosine gatekeeper residue was likely to be the reason why the APH(2 00 )-Ia enzyme was an exclusively GTPutilizing phosphotransferase (Bhattacharya et al, 2013). In APH(2 00 )-IIa, although the gatekeeper Met85 [APH(2 00 )-IIa residue numbering] side chain projects into the binding cleft, it does not interfere with the positioning of an adenine.…”

“…In APH(2 00 )-IIa, although the gatekeeper Met85 [APH(2 00 )-IIa residue numbering] side chain projects into the binding cleft, it does not interfere with the positioning of an adenine. In APH(2 00 )-IVa the gatekeeper Phe95 [APH(2 00 )-IVa residue numbering] is in a different conformation which has the phenyl ring in an adjacent secondary hydrophobic pocket (Bhattacharya et al, 2013;Toth et al, 2010).…”