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

Smith, Clyde A.; Tóth, Márta; Frase, Hilary; Byrnes, Laura J.; Vakulenko, Sergei B.

doi:10.1074/jbc.m112.341206

Cited by 24 publications

(45 citation statements)

References 59 publications

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“…Modeling of a tyrosine side chain into the APH(2Љ)-IIa secondary pocket shows that it severely clashes with the bulky F57 side chain. Therefore, it is predicted, based upon in silico modeling, that a tyrosine side chain introduced at this position would project out into the nucleotide-binding pocket in much the same way as that observed in the wild-type APH(2Љ)-IIIa structure (14) and thus would effectively block the efficient binding of an ATP molecule, making the enzyme unable to utilize ATP as a cosubstrate, as shown by the kinetic data. These structural analyses suggest that both the size of the "gatekeeper" side chain and its ability to move away from the NTP-binding site control access to the inner ATP-binding template of aminoglycoside 2Љ-phosphotransferases and thus determine their cosubstrate specificity.…”

Section: Resultsmentioning

confidence: 91%

“…Recent structural studies of the APH(2Љ)-IIa, -IIIa, and -IVa phosphotransferases have provided detailed information on the architecture of their NTP-binding sites and allowed us to explain the nucleotide specificity of these aminoglycoside kinases (12)(13)(14)19). The dual NTP specificity of APH(2Љ)-IIa and APH(2Љ)-IVa results from the existence in their nucleotide-binding sites of distinct but overlapping structural templates for the binding of ATP and GTP, with the ATP-binding template located deeper in the nucleotidebinding pocket (12,13,19).…”

Section: Resultsmentioning

confidence: 99%

“…The dual NTP specificity of APH(2Љ)-IIa and APH(2Љ)-IVa results from the existence in their nucleotide-binding sites of distinct but overlapping structural templates for the binding of ATP and GTP, with the ATP-binding template located deeper in the nucleotidebinding pocket (12,13,19). In APH(2Љ)-IIIa, the ATP-binding template is blocked by a bulky tyrosine residue ("gatekeeper" residue), resulting in an inability of the enzyme to utilize ATP as a cosubstrate (14). Replacement of this bulky "gatekeeper" residue by alanine broadens the NTP specificity of APH(2Љ)-IIIa by allowing the enzyme to utilize both ATP and GTP (14).…”

Section: Resultsmentioning

confidence: 99%

“…In APH(2Љ)-IIIa, the ATP-binding template is blocked by a bulky tyrosine residue ("gatekeeper" residue), resulting in an inability of the enzyme to utilize ATP as a cosubstrate (14). Replacement of this bulky "gatekeeper" residue by alanine broadens the NTP specificity of APH(2Љ)-IIIa by allowing the enzyme to utilize both ATP and GTP (14).…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Bhattacharya

Tóth

Smith

et al. 2013

Antimicrob Agents Chemother

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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 ...

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Bhattacharya

Tóth

Smith

et al. 2013

Antimicrob Agents Chemother

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Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains

Parulekar

Sonawane

2017

J of Cellular Biochemistry

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Antibiotic resistance to aminoglycoside group of antibiotics mainly occurs by aminoglycoside kinases (AKs). Thus, targeting AKs from different multidrug resistant (MDR) strains could result into inhibition of AK enzymes and ultimately the resistance. Therefore, the present study aims to target one of these AKs that is APH(3')-Ia from Acinetobacter baumannii through structure based virtual screening (SBVS) and test the binding affinity of the most stable virtually screened inhibitor with AKs from Mycobacterium tuberculosis, Acinetobacter baumannii, Enterococcus gallinarum, and Escherichia coli. SBVS investigated ZINC71575479 as a most stable inhibitor with -8.92 kcal/mol of binding energy and 0.66 µM of inhibition constant. Molecular docking results revealed that the ZINC71575479 can efficiently bind to nucleotide triphosphate (NTP) binding site of different AKs which is a known drug target site. Sequence analysis study of different AKs showed no significant similarity for active site residues; however structure superimposition study showed conserved NTP-binding domain. Molecular dynamics (MD) simulations showed stable behavior of all docked complexes with notable conformational stability of salt bridges at NTP-binding site of different AKs. Binding energy calculations revealed the interactions between key residues from NTP- binding domain of different AKs with ZINC71575479. In order to validate the MD simulations and binding energy results, crystal structure complexed with tyrphostin AG1478 a known inhibitor of AKs was kept as control. Thus, this work demonstrates the binding efficiency of ZINC71575479 toward different AKs for circumventing aminoglycoside resistance and opens avenues for the development of new antibiotics that can target diverse MDR strains with aminoglycoside resistance.

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Insights into the antibiotic resistance and inhibition mechanism of aminoglycoside phosphotransferase from Bacillus cereus: In silico and in vitro perspective

Parulekar

Sonawane

2018

J of Cellular Biochemistry

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Because of the lack of structural studies on aminoglycoside phosphotransferase (APH) from prevalent volatile human pathogen Bacillus cereus, aminoglycoside resistance therapeutics research remains elusive. Hence, in this computational study, we have performed homology modeling, molecular docking, molecular dynamics (MD), and principal component analysis studies on APH from B. cereus. The structure of APH was predicted by homology modeling using MODELLER 9v12 and validated for its stereochemical qualities. Sequence analysis study of the template (Protein Data Bank ID: 3TDW) and APH from B. cereus sensu lato group showed exact matching of active-site residues. The mechanism of substrate and inhibitor binding to APH was studied using molecular docking, which identified GTP as the more preferred substrate, whereas ZINC71575479 as the most effective inhibitor. The active-site residues, ARG41, TYR90, ASP195, and ASP215 at nucleotide triphosphate-binding cavity of APH were found to be involved in binding with substrate and inhibitor. Molecular dynamics simulation study of APH in apo form and bound form confirmed the stability and effective binding of GTP and ZINC71575479 in a dynamic state. Molecular mechanics Poisson-Boltzmann surface area calculations revealed energetic contributions of active-site residues of APH in binding with GTP and ZINC71575479. The principal component analysis revealed the internal global motion of APH in apo and complex form. Furthermore, experimental studies on APH from B. cereus ATCC 10876 validated the in silico findings for its inhibition. Thus, this study provides more information on structure-function relationships of APH from B. cereus and open avenues for designing effective strategies to overcome antibiotic resistance.

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

Cited by 24 publications

References 59 publications

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

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

Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains

Insights into the antibiotic resistance and inhibition mechanism of aminoglycoside phosphotransferase from Bacillus cereus: In silico and in vitro perspective

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