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Literature summary extracted from

  • Smith, C.; Toth, M.; Stewart, N.; Maltz, L.; Vakulenko, S.
    Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases (2019), Acta Crystallogr. Sect. D, 75, 1129-1137 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.190 gene aphA, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Enterococcus sp.

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.1.190 ternary complex of APH(2'')-IIIa with GDP and kanamycin, the ternary complex s prepared by adding a tenfold molar excess of Mg2-GTP and kanamycin to the apo APH(2'')-IIIa F108L enzyme, followed by incubation of the complex at 4x02C for 2 h, the complex is crystallized from 30% PEG 4000, and 0.1 M Tris-HCl pH 8.5, X-ray diffraction structure determination at 1.34 A resolution, molecular replacement using the binary Mg2-GDP-APH(2'')-IIIa complex as the starting model (PDB ID 3tdw) Enterococcus sp.

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.190 F108L site-directed mutagenesis, the point mutant and wild-type enzymes have the same structure Enterococcus sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.7.1.190 additional information the ternary complex between APH(2'')-IIIa, GDP and kanamycin can be regarded as an inactive abortive complex, since the gamma-phosphate group which would normally be transferred to the 2''-hydroxyl of the substrate is absent. The cofactor binding in the ternary complex is similar in detail to that in the previously described binary complex Enterococcus sp.

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.190 Mg2+ required Enterococcus sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.190 GTP + kanamycin Enterococcus sp.
-
 GDP + kanamycin 2''-phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.190 Enterococcus sp.
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.190 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by kanamycin and gentamicin affinity chromatography Enterococcus sp.

Reaction

EC Number Reaction Comment Organism Reaction ID
2.7.1.190 GTP + gentamicin = GDP + gentamicin 2''-phosphate mechanism of the phosphorylation of aminoglycoside antibiotics by APH(2'') enzymes, detailed overview Enterococcus sp.
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.190 GTP + kanamycin
-
 Enterococcus sp. GDP + kanamycin 2''-phosphate
-
 ?
2.7.1.190 additional information substrate binding in the APH(2'') enzymes, overview. The substrate molecules are bound in essentially the same orientation in all structures. The neamine moiety of aminoglycosides is nestled against the core subdomain and helix alpha9 such that the A ring projects towards motif 3 in the helical subdomain and the B ring points towards motif 1 and motif 2 in the core subdomain. The C ring then projects back towards the helical domain Enterococcus sp. ?
-
-

Synonyms

EC Number Synonyms Comment Organism
2.7.1.190 aminoglycoside phosphotransferase
-
 Enterococcus sp.
2.7.1.190 aminoglycoside-2''-phosphotransferase
-
 Enterococcus sp.
2.7.1.190 APH
-
 Enterococcus sp.
2.7.1.190 APH(2'')-IIIa
-
 Enterococcus sp.

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.190 GTP
-
 Enterococcus sp.

General Information

EC Number General Information Comment Organism
2.7.1.190 evolution aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification, the APH(2'') family comprises four distinct members Enterococcus sp.
2.7.1.190 additional information structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2''-phosphotransferases. Structure comparisons of the ternary complex of APH(2'')-IIIa with GDP and kanamycin with substrate-bound structures of APH(2'')-Ia, APH(2'')-IIa and APH(2'')-IVa. In contrast to the case for APH(2'')-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2'')-IIa, APH(2'')-IIIa and APH(2'')-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes. The ternary complex between APH(2'')-IIIa, GDP and kanamycin can be regarded as an inactive abortive complex, since the gamma-phosphate group which would normally be transferred to the 2''-hydroxyl of the substrate is absent. The cofactor binding in the ternary complex is similar in detail to that in the previously described binary complex Enterococcus sp.