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 - PubMed (original) (raw)

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

Paul G Young et al. J Bacteriol. 2009 Jul.

Abstract

Aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2'')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2'')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-(beta,gamma-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2'') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2'')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R(free), 0.271) at a resolution of 2.5 A. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2'')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2'') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

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Figures

FIG. 1.

FIG. 1.

Structures of gentamicin, kanamycin, streptomycin, and neomycin. Gentamicin and kanamycin are classified as 4,6-disubstituted aminoglycosides, whereas neomycin is an example of a 4,5-disubstituted compound. The three structural variants which comprise gentamicin C are indicated. Amikacin is similar to kanamycin, although the substituent on the N1 amine is a 4-amino-2-hydroxy-1-oxobutyl group. Taken together, the A and B rings of aminoglycosides, such as gentamicin, kanamycin, and neomycin, are commonly known as the neamine moiety.

FIG. 2.

FIG. 2.

Crystal structure of APH(2′′)-IIa. (a) Stereo view of the APH(2′′)-IIa monomer showing the location of the substrate binding site, indicated by a gentamicin molecule in yellow, and the nucleotide binding site, indicated by a partially transparent AMP molecule (upper panel). The N-terminal domain is at the top of the figure (pale green), with the core subdomain in the center (bright green) and the helical subdomain at the bottom (dark green). The numbering of the secondary structure elements is indicated. (b) Stereo view of the superposition of the gentamicin-APH(2′′)-IIa complex (green ribbons) and the kanamycin-APH(3′)-IIa complex (red ribbons) based upon residues from the core subdomain. The similarity of the N domain and the core subdomain can be seen, along with the differences in the helical subdomain. The location of the substrate binding site is indicated by a yellow gentamicin molecule and a kanamycin molecule in thinner white sticks. (c) Stereo view of the close-up view of the area inside the black box in panel b, showing the substrate binding site with the “top” and the “base” of the binding cleft indicated. The three rings of the gentamicin (yellow sticks) are labeled in boldface type (C-B-A). The kanamycin molecule as it is bound to APH(3′)-IIa is shown as thin white sticks with the three rings labeled in italics (_A_-_B_-C).

FIG. 3.

FIG. 3.

Gentamicin binding site. (a) Stereo view of gentamicin in the binary gentamicin-APH(2′′)-IIa complex showing composite omit 2_Fo_-Fc electron density (pink; 1.0 σ) for the gentamicin substrate (yellow ball-and-stick representation). The rings are labeled in boldface type (C-B-A). The hydrogen bonding interactions with the substrate are indicated as dashed lines. The kinked helix α9 is at the lower right. The final 2_Fo_-Fc density (blue; 1.0 σ) is shown for some of the protein residues. (b) View in panel a rotated vertically by 90° and showing the gentamicin bound in the refined position (yellow ball-and-stick representation) and in an alternative “upside-down” orientation (thinner gray sticks). The rings are labeled in boldface type for the correct orientation (C-B-A), and for the alternative orientation, only the A and C rings are labeled in gray italics for clarity.

FIG. 4.

FIG. 4.

Binding sites in the ternary complex. (a) Stereo view of streptomycin in the ternary AMPPCP-streptomycin-APH(2′′)-IIa complex showing composite omit 2_Fo_-Fc density (pink; 1.0 σ) for the streptomycin inhibitor from molecule A (cyan ball-and-stick representation). The equivalent streptomycin conformation from molecule B is overlaid as thin bonds (gray). The location of helices α9 and α10 from the binary gentamicin-APH(2′′)-IIa complex are shown as semitransparent gray coils. (b) Stereo view of the AMPPCP (gray ball-and-stick representation) in the ternary AMPPCP-streptomycin-APH(2′′)-IIa complex showing composite omit 2_Fo_-Fc density (pink; 1.0σ). The magnesium ion is shown as a yellow sphere surrounded by the chelating AMPPCP triphosphate moiety. The hydrogen-bonding interactions with the AMPPCP are indicated by dashed black lines. Final 2_Fo_-Fc densities (blue; 1.0 σ) for surrounding protein residues are shown, along with the 2_Fo_-Fc density for the interdomain linker (gold bonds, left).

FIG. 5.

FIG. 5.

Surface representation of the substrate binding site in APH(2′′)-IIa (green) showing the bound gentamicin (yellow sticks) and an AMPPNP molecule (colored sticks; top) from APH(3′)-IIIa modeled into the structure based upon the superposition of the catalytic aspartate loop [residues 189 to 197 in APH(2′′)-IIa; 187 to 195 in APH(3′)-IIIa]. (a) The kanamycin from APH(3′)-IIa is shown as thin gray bonds. APH(3′)-IIa was superimposed on APH(2′′)-IIa based upon the catalytic aspartate loop. The location of this residue (Asp192) is indicated. When the structures are superimposed in this way, the hydroxyl group which is phosphorylated on both substrates is in essentially the same position (indicated by the black circle). (b) The kanamycin molecule modeled in the same conformation as gentamicin. In this orientation, the 2′′-OH of the kanamycin overlaps the equivalent atom on gentamicin (black circle). (c) The superimposed streptomycin from molecule B of the ternary AMPPCP-streptomycin-APH(2′′)-IIa complex, shown as thin gray sticks. The streptomycin occupies approximately the same location as the gentamicin. One of the guanidinium groups on streptomycin A ring projects upwards toward the nucleotide binding site and would clearly interfere with the β- and γ-phosphate groups of an extended ATP triphosphate. (d) A neomycin molecule modeled in the gentamicin binding site such that the A and B rings occupy approximately the same position as the corresponding rings in gentamicin. The C projects toward the helical subdomain, and in this orientation, the D ring of neomycin is hidden behind the helical subdomain.

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