Structural basis for the O- acetyltransferase function of the extracytoplasmic domain of OatA from Staphylococcus aureus

Abstract

Many bacteria possess enzymes that modify the essential cell-wall polymer peptidoglycan by O-acetylation. This modification occurs in numerous Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, a common cause of human infections. O-Acetylation of peptidoglycan protects bacteria from the lytic activity of lysozyme, a mammalian innate immune enzyme, and as such is important for bacterial virulence. The O-acetylating enzyme in Gram-positive bacteria, O-acetyltransferase A (OatA), is a two-domain protein consisting of an N-terminal integral membrane domain and a C-terminal extracytoplasmic domain. Here, we present the X-ray crystal structure at 1.71 Å resolution and the biochemical characterization of the C-terminal domain of S. aureus OatA. The structure revealed that this OatA domain adopts an SGNH-hydrolase fold and possesses a canonical catalytic triad. Site-specific replacement of active-site amino acids revealed the presence of a water-coordinating aspartate residue that limits esterase activity. This residue, although conserved in staphyloccocal OatA and most other homologs, is not present in the previously characterized streptococcal OatA. These results provide insights into the mechanism of acetyl transfer in the SGNH/GDSL hydrolase family and highlight important evolutionary differences between homologous OatA enzymes. Furthermore, this study enhances our understanding of PG O-acetyltransferases, which could guide the development of novel antibacterial drugs to combat infections with multidrug-resistant bacterial pathogens.

Keywords: O-acetylation; O-acetyltransferase; OatA; Staphylococcus aureus (S. aureus); X-ray crystal structure; bacterial cell wall; cell wall; methicillin-resistant Staphylococcus aureus (MRSA); peptidoglycan; virulence factor.

Figure 1.

Structure of SaOatA_C. A, cartoon representation of SaOatA_C showing seven α-helices (white) and five β-sheets (blue) arranged in an α/β-hydrolase fold. The putative catalytic triad residues are illustrated as orange sticks. The sodium atom located between α-helix 4 and β-sheet 5 is indicated by a purple sphere. B, surface representation of SaOatA_C showing the putative catalytic triad residues in orange in a shallow active-site depression.

Figure 2.

Structural comparison of SaOatA_C to SGNH/GDSL hydrolase homologs. A, cartoon representation of SaOatA_C (dark blue; PDB code 6VJP) overlaid with SpOatA_C (pink; PDB code 5UFY), Axe2 from G. stearothermophilus (GsAxe2; gray; PDB code 4JHL), family 3 carbohydrate esterase from C. thermocellum (CtCes3; yellow; PDB code 2VPT), and family 3 carbohydrate esterase from T. cellulolyticus (TcCes3; pale blue; PDB code 5B5S). B, alignment of the conserved SGNH hydrolase block residues from the aforementioned structural homologs. C, block alignments comparing SaOatA, SpOatA, and SGNH hydrolase structural homologs. Catalytic triad and oxyanion hole residues are in red and blue, respectively, whereas the highly conserved Asp present in most OatA sequences except those of the streptococci node is in green. SaOatAC, PDB ID: 6VJP; SpOat-AC, PDB ID: 5UFY, RMSD: 1.8 Å over 179 equivalent Cα's, z-score: 20, 27% sequence identity; GsAxe2, PDB ID: 4JHL, RMSD: 2.2 Å over 146 equivalent Cα's, z-score: 16.5, 18% sequence identity; CtCes3, PDB ID: 2VPT, RMSD: 2.2 Å over 147 equivalent Cα's, z-score: 16.7, 14% sequence identity; TcCes3, PDB ID: 5B5S over 149 equivalent Cα's, RMSD: 2.2 Å, z-score: 16.4, 17% sequence identity.

Figure 3.

Direct observation of the acetyl-SaOatA_C intermediate. A, SaOatA_C was incubated with 1 mm pNP-Ac (red) or no substrate (black) and separated via reverse-phase LC-MS. The resulting mass spectra show the appearance of a peak with a mass increase of 42 Da after incubation with pNP-Ac, consistent with the formation of a covalent acetyl-enzyme intermediate. B, table showing the expected and observed ions for the fragmentation of an ion from a trypsin-digested reaction of SaOatA_C with pNP-Ac. A reaction of SaOatA_C with 1 mm pNP-Ac was quenched by the addition of cold acetone. The recovered protein was digested with trypsin, and the resultant peptides were separated by LC-MS/MS. The parent ion had an m/z of 1138.7, corresponding to the amino acid sequence shown. The fragmentation pattern was consistent with the acetylation of Ser⁴⁵³ (shown in red). The observed ions are noted in green or blue.

Figure 4.

The active site of SaOatA_C. A water molecule (red sphere) can be seen coordinated by the Oδ1 of Asp⁴⁵⁷, the backbone carbonyl of Ile⁵⁷⁷, and the Oγ of the catalytic Ser⁴⁵³.