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Literature summary for 2.7.1.190 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)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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

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

Inhibitors

Inhibitors Comment Organism Structure
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

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Enterococcus sp.
-
-
-

Purification (Commentary)

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

Reaction

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
GTP + kanamycin
-
Enterococcus sp. GDP + kanamycin 2''-phosphate
-
?
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

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

Cofactor

Cofactor Comment Organism Structure
GTP
-
Enterococcus sp.

General Information

General Information Comment Organism
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.
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.