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Literature summary extracted from

  • Caldwell, S.J.; Huang, Y.; Berghuis, A.M.
    Antibiotic binding drives catalytic activation of aminoglycoside kinase APH(2'')-Ia (2016), Structure, 24, 935-945 .
    View publication on PubMed

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.1.190 purified recombinant wild-type and mutant enzymes in complex with cofactor GTP, GDP, and especially GMPPNP, and substrates gentamycin C1, rbiostamycin, kanamycin A, neomycin B in diffenrent combinations, X-ray diffraction structure determination and analysis at 2.15-2.50 A resolution Staphylococcus aureus

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.190 S214A site-directed mutagenesis Staphylococcus aureus
2.7.1.190 Y237F site-directed mutagenesis, the mutant removes the other hydrogen bond between the phosphate and the protein, and a greatly reduced electron density for the gamma-phosphate is observed Staphylococcus aureus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.190 Mg2+ required, magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules Staphylococcus aureus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.190 GTP + gentamicin C1 Staphylococcus aureus
-
 GDP + gentamicin C1 2''-phosphate
-
 ?
2.7.1.190 GTP + kanamycin A Staphylococcus aureus
-
 GDP + kanamycin A 2''-phosphate
-
 ?
2.7.1.190 GTP + neomycin B Staphylococcus aureus
-
 GDP + neomycin B 2''-phosphate
-
 ?
2.7.1.190 GTP + ribostamycin Staphylococcus aureus
-
 GDP + ribostamycin 2''-phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.190 Staphylococcus aureus P0A0C1
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.190 GTP + gentamicin C1
-
 Staphylococcus aureus GDP + gentamicin C1 2''-phosphate
-
 ?
2.7.1.190 GTP + kanamycin A
-
 Staphylococcus aureus GDP + kanamycin A 2''-phosphate
-
 ?
2.7.1.190 GTP + neomycin B
-
 Staphylococcus aureus GDP + neomycin B 2''-phosphate
-
 ?
2.7.1.190 GTP + ribostamycin
-
 Staphylococcus aureus GDP + ribostamycin 2''-phosphate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.7.1.190 AAC(6')-Ie/APH(2'')-Ia
-
 Staphylococcus aureus
2.7.1.190 aac6-aph2
-
 Staphylococcus aureus
2.7.1.190 aacA-aphD
-
 Staphylococcus aureus
2.7.1.190 aminoglycoside kinase
-
 Staphylococcus aureus
2.7.1.190 APH(2'')-Ia
-
 Staphylococcus aureus
2.7.1.190 bifunctional aminoglycoside acetyltransferase/phosphotransferase
-
 Staphylococcus aureus

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.190 GTP binding structure Staphylococcus aureus
2.7.1.190 additional information cofactor binding structures, conformations of triphosphate group in structures of APH(2'')-Ia with GTP, GDP, and GMPPNP, overview. Two hydrogen bonds are formed between the triphosphate and residues S214 of the Gly loop and Y237. The nucleoside cosubstrates bind in the cleft between the N lobe (residues 180-279) and C lobe (residues 280-479), and two magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules Staphylococcus aureus

General Information

EC Number General Information Comment Organism
2.7.1.190 additional information the open-closed transition in APH(2'')-Ia brings distal regions of the protein into contact. The enzyme also exhibits a novel phenomenon: a switch between two welldefined triphosphate conformations. Interactions between the helical subdomain and N lobe loops connect enzyme closure to triphosphate activation. APH(2'')-Ia open-closed transition links aminoglycoside binding to catalysis through the Gly loop. In the stabilized conformation, the enzyme does not form most of the interactions that are required for catalysis in kinases. The gamma-phosphate does not coordinate between the two catalytic magnesium ions. There is no catalytic base in position to activate the incoming nucleophile, and no positively charged residue in place to stabilize the leaving group. To hydrogen bonds are formed between the triphosphate and residues S214 of the Gly oop and Y237. Aminoglycoside molecules bind in the cleft between the core (residues 280-322 and 366-432) and helical (residues 322-365 and 433-479) subdomains of the C lobe, the nucleoside cosubstrates bind in the cleft between the N lobe (residues 180-279) and C lobe (residues 280-479), and two magnesium ions, Mg1 and Mg2, are consistently resolved with coordinating water molecules. Aminoglycosides bind to APH(2'')-Ia via conserved rings, while variable rings Dictate reactivity Staphylococcus aureus
2.7.1.190 physiological function APH(2'')-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2'')-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using GTP as phosphate donor Staphylococcus aureus