Potential for reduction of streptogramin A resistance revealed by structural analysis of acetyltransferase VatA

Abstract

Combinations of group A and B streptogramins (i.e., dalfopristin and quinupristin) are "last-resort" antibiotics for the treatment of infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Resistance to streptogramins has arisen via multiple mechanisms, including the deactivation of the group A component by the large family of virginiamycin O-acetyltransferase (Vat) enzymes. Despite the structural elucidation performed for the VatD acetyltransferase, which provided a general molecular framework for activity, a detailed characterization of the essential catalytic and antibiotic substrate-binding determinants in Vat enzymes is still lacking. We have determined the crystal structure of S. aureus VatA in apo, virginiamycin M1- and acetyl-coenzyme A (CoA)-bound forms and provide an extensive mutagenesis and functional analysis of the structural determinants required for catalysis and streptogramin A recognition. Based on an updated genomic survey across the Vat enzyme family, we identified key conserved residues critical for VatA activity that are not part of the O-acetylation catalytic apparatus. Exploiting such constraints of the Vat active site may lead to the development of streptogramin A compounds that evade inactivation by Vat enzymes while retaining binding to their ribosomal target.

FIG 1

Overall structure of VatA. (A) Chemical structures of streptogramin A compounds virginiamycin M1 and dalfopristin. The O-18 arrows indicate the sites of O-acetylation. (B) Top, domain architecture (cap, LβH, α domain, C-terminal/CT) of the three chains of trimeric VatA. Bottom, structure of Vat, colored according to the top domain architecture diagram. The ternary complex shown was modeled by the superposition of the crystal structures of the binary complexes of VatA-virginiamycin M1 and VatA-acetyl-CoA. Virginiamycin M1 and acetyl-CoA are shown in a stick representation. Yellow stars indicate the locations of the catalytic centers. The subscripts after the domain names, acetyl-CoA ligands, or termini (N, N terminus; C, C terminus) indicate the protein chain. (C) Superposition of VatA and VatD structures (8, 9). The VatA chains are colored in red, blue, and green, and all chains of VatD are colored dark gray.

FIG 2

Sequence analysis of Vat and closely related enzymes. (A) Multiple-sequence alignment. The sequence names are colored by groups as defined below, and the numbers refer to NCBI gi numbers. The domain architecture is indicated above the sequences. VatA residues that contacted an antibiotic (virginiamycin M1) are labeled. (B) Maximum likelihood phylogenetic tree constructed from alignment. The cluster 1 and 2 Vat enzymes are highlighted in red and blue, respectively. The bootstrap percentage is indicated at each node. The arrows refer to the tree root, and the dashed line indicates the branches for the outgroup sequences, which exceed the size of the page. The scale bar represents 0.2 substitutions per site.

FIG 3

Active sites of VatA. (A) Streptogramin A binding site of VatA. VatA is shown in a surface representation and colored as in Fig. 1A. Virginiamycin M1 is shown in sticks. The white dashed box is the location of the acetyl-CoA-binding tunnel. (B) Details of interactions between VatA and virginiamycin M1. The dashes indicate hydrogen bonds. The electron density for virginiamycin M1 shown is a simulated omit Fo–Fc density contoured at 1.0 σ. (C) Acetyl-CoA binding site of VatA. VatA is shown in a surface representation and colored as in Fig. 1A. The yellow dashed box is the location of the virginiamycin M1 binding site. (D) Details of interactions between VatA and acetyl-CoA. The dashes indicate hydrogen bonds. The electron density for acetyl-CoA shown is a simulated omit Fo–Fc density contoured at 2.0 σ.

FIG 4

Comparison of ligand binding to VatA and VatD. (A) Comparison of interactions between VatA and virginiamycin M1 (Virg. M1) with VatD (8, 9) and virginiamycin M1 or dalfopristin. Two views are shown, rotated 80°. The enzyme side chains are colored as follows: purple and green for chain A and chain C of VatA, gray for both chains A and B of VatD; side chains from the VatD-dalfopristin complex are not shown, as no major conformational differences are observed with the VatD-virginiamycin M1 complex. Virginiamycin M1 bound to VatA and VatD is colored in yellow and light gray, respectively, and dalfopristin bound to VatD is colored in black. (B) Comparison of acetyl-CoA binding to VatA (three copies per trimer, colored purple) and VatD (three copies per trimer [9], colored gray). The circle highlights the distinct conformation of acetyl and β-mercaptoethylamine groups. (C) Comparison of interactions between acetyl-CoA and VatA and VatD, colored the same as in panel A.

FIG 5

Acetylation activity of VatA and mutants against streptogramin A compounds. Shown is a comparison of the acetylation activity of VatA WT and mutants toward virginiamycin M1 (black bars) and dalfopristin (white bars). The specific activity of each enzyme is the average from the data of three independent experiments.

FIG 6

(A) Critical contacts between Vat enzymes and streptogramin A. Side chains from the VatA active site and virginiamycin M1 atoms are colored according to combined sequence and mutational analysis: darker blue, most critical for acetylation reaction, fully conserved; lighter blue, also important for acetylation reaction, highly conserved within amino acid class; red, critical for acetylation reaction, conserved within Vat enzyme clusters only; gray, positions tolerant to mutation, not well conserved. The hatched residues were not mutated. The distance labels are the distances from those particular side chain atoms to the acetylation site of virginiamycin M1 (O-18). (B) Putative catalytic mechanism of VatA.