The Crystal Structures of Substrate and Nucleotide Complexes of
            <i>Enterococcus faecium</i>
            Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

Young, Paul G.; Walanj, Rupa; Lakshmanan, Venkatachalam; Byrnes, Laura J.; Metcalf, Peter; Baker, Edward N.; Vakulenko, Sergei B.; Smith, Clyde A.

doi:10.1128/jb.00149-09

Cited by 52 publications

(72 citation statements)

References 54 publications

(56 reference statements)

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“…This is similar to that described for the equivalent helix in APH(2 00 )-IIa. 18 The first half of this helix matches well in the two crystal forms but beyond Trp271 the form II helix a9 becomes slightly less kinked and subsequently deviates from the path of the helix in form I. This, in turn, puts helix a10 in a different position (Supporting Information Figure S2B).…”

Section: Kinetic Mechanismmentioning

confidence: 70%

“…Residues that are involved in gentamicin binding in APH(2 00 )-IIa, and implicated in substrate binding in APH(2 00 )-IVa, are all completely conserved in the APH(2 00 )-Ia and APH(2 00 )-IIIa enzymes. 18 This implies that the substrate-binding sites in the four APH(2 00 ) phosphotransferases might be similar, with small changes to account for the observed differences in their resistance profiles. Further analysis of these differences must await the elucidation of the structures of the remaining two enzymes.…”

Section: Aph(2 00 )-Mediated Aminoglycoside Resistancementioning

confidence: 99%

“…The related APH(2 00 )-IIa structure 18 Figure S2A) shows that although the Nterminal and core subdomains appear to be in roughly the same relative orientations, three of the helices (a5, a6, and a10) in the helical subdomain are in a different position in the two structures. When the helical subdomain is removed from the superposition calculation, the N-terminal domain and the core subdomains match significantly better with an RMSD of 0.5 Å for 207 matching C a atoms.…”

Section: Kinetic Mechanismmentioning

confidence: 99%

“…17 We recently reported the structure of the first APH(2 00 ) enzyme, both as the binary gentamicin-APH(2 00 )-IIa complex and the ternary complex containing b,c-methylene adenosine triphosphate (AMPPCP) and the inhibitor streptomycin. 18 The enzyme folds into two structural domains, an N-terminal b-sheet domain comprising~100 residues, and a larger C-terminal domain composed predominantly of a-helices. The ATP-binding site is sandwiched between the N-terminal and C-terminal domains, with the aminoglycoside substrate-binding site located in a cleft in the C-terminal domain.…”

Section: Introductionmentioning

confidence: 99%

“…There are three additional aminoglycoside phosphotransferase structures, APH(9) (spectinomycin phosphotransferase) 11 and two from the APH(3 0 ) family of enzymes, 19,20 and despite a very low sequence identity between these APH(9), APH(3 00 ), and APH(2 00 ) families, there is a marked degree of structural similarity. 11,18 Here, we report the X-ray structure, substrate profile, and kinetic mechanism of a second member of the APH(2 00 ) subfamily, the APH(2 00 )-IVa aminoglycoside phosphotransferase, and for the first time we report the determinants of NTP recognition by these clinically important enzymes.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2010

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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.

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Section: Kinetic Mechanismmentioning

confidence: 70%

Section: Aph(2 00 )-Mediated Aminoglycoside Resistancementioning

confidence: 99%

Section: Kinetic Mechanismmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2010

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Overcoming Multidrug‐Resistant MRSA Using Conventional Aminoglycoside Antibiotics

et al. 2020

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Global multidrug‐resistant (MDR) bacteria are spreading rapidly and causing a great threat to human health due to the abuse of antibiotics. Determining how to resensitize MDR bacteria to conventional inefficient antibiotics is of extreme urgency. Here, a low‐temperature photothermal treatment (PTT, 45 °C) is utilized with red phosphorus nanoparticles to resensitize methicillin‐resistant Staphylococcus aureus (MRSA) to conventional aminoglycoside antibiotics. The antibacterial mechanism is studied by the proteomic technique and molecular dynamics (MD) simulation, which proves that the aminoglycoside antibiotics against MRSA can be selectively potentiated by low‐temperature PTT. The catalytic activity of 2‐aminoglycoside phosphotransferase (APH (2″))—a modifying enzyme—is demonstrated to be obviously inhibited via detecting the consumption of adenosine triphosphate (ATP) in the catalytic reaction. It is also found that the active site of aspartic acid (ASP) residues in APH (2″) is thermally unstable from the results of molecular dynamics simulation. Its catalytic ability is inhibited by preventing the deprotonating procedure for the target OH of gentamycin. The combined therapy also exhibits great biocompatibility and successfully treats MRSA infections in vivo. This low‐temperature PTT strategy has the potential to be an exogenous‐modifying enzyme inhibitor for the treatment of MDR bacterial infection.

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The Future of Aminoglycosides: The End or Renaissance?

et al. 2010

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Although aminoglycosides have been used as antibacterials for decades, their use has been hindered by their inherent toxicity and the resistance that has emerged to these compounds. It seems that such issues have relegated a formerly front-line class of antimicrobials to the proverbial back shelf. However, recent advances have demonstrated that novel aminoglycosides have a potential to overcome resistance as well as to be used to treat HIV-1 and even human genetic disorders, with abrogated toxicity. It is not the end for aminoglycosides, but rather, the challenges faced by researchers have led to ingenuity and a change in how we view this class of compounds, a renaissance.

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The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

Cited by 52 publications

References 54 publications

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

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

Overcoming Multidrug‐Resistant MRSA Using Conventional Aminoglycoside Antibiotics

The Future of Aminoglycosides: The End or Renaissance?

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