Structural Analyses of Nucleotide Binding to an Aminoglycoside Phosphotransferase^,

Abstract

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.

Small-Angle X-Ray Scattering Analysis of the Bifunctional Antibiotic Resistance Enzyme Aminoglycoside (6′) Acetyltransferase-Ie/Aminoglycoside (2″) Phosphotransferase-Ia Reveals a Rigid Solution Structure

“…This behavior is similar to what has been observed for APH(3=)-IIIa, another clinically important aminoglycoside resistance enzyme (6), but is in sharp contrast to what is seen for APH(9)-Ia, an enzyme whose role in antibiotic resistance is debatable (17). We have previously suggested that the absence of major conformational changes may signify that the aminoglycoside resistance enzyme is optimized for rapid drug detoxification and, thus, is an important feature of dedicated resistance enzymes (6). This would clearly be consistent with the existence of AAC(6=)-Ie/APH(2Љ)-Ia in numerous clinical isolates (10).…”

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

“…Structural details are currently known for only two members of the APH(3Ј) family, APH(3Ј)-IIIa (5,18,23) and APH(3Ј)-IIa (37). These enzymes share a two-domain structure similar to the catalytic domains of the eukaryotic Ser/Thr and Tyr protein kinases.…”

Role ofptsP,orfT, andsssRecombinase Genes in Root Colonization byPseudomonas fluorescensQ8r1-96

“…OrfT is a member of COG3178, which contains predicted phosphotransferases (E value, 1e Ϫ90 ), and further analyses revealed the presence of a Pfam domain associated with the aminoglycoside phosphotransferase enzyme family (residues 152 to 234). The OrfT structure predicted by 3D-PSSM (29) is related to the structures of aminoglycoside 3Ј-phosphotransferases from Enterococcus faecalis (10) and Klebsiella pneumoniae (45).…”

“…pisi. Strain Q8r1-96 is exceptional because of its ability to establish and maintain large populations (up to 10 7 CFU/g of root) on the roots of wheat, pea (34,51), and sugar beet (6) even when low doses are used. This special colonizing ability is typical of all D-genotype strains tested to date (34,35,51) and distinguishes these strains from typical rhizosphere pseudomonads.…”