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.

Introduction

Antimicrobial resistance is one of the leading healthcare burdens of the century and is only predicted to worsen. Current projections predict that antimicrobial resistant infections will overtake cancer as a leading cause of death worldwide by 2050 (1). Among the biggest threats is methicillin-resistant Staphylococcus aureus. Individuals infected with methicillin-resistant S. aureus are estimated to be 64% more likely to die than those infected with non–drug-resistant strains of S. aureus. Both the Centers for Disease Control and Prevention and the World Health Organization have highlighted the desperate need for the research and development of novel antimicrobials to combat multidrug-resistant infections (2, 3). One approach researchers are taking is to search for ways to disarm bacteria with nontraditional therapeutic agents (4). By targeting virulence factors that significantly contribute to the ability of a bacterium to colonize a host or cause infection, it will be possible to prevent infection without otherwise affecting survivability of the bacterium. It is thought that drugs targeting virulence factors may suffer less from the development of resistance (4).

The peptidoglycan (PG) layer of Gram-negative and Gram-positive bacteria is an essential component of the cell envelope involved in shape determination and resisting turgor pressure. PG is composed of a glycan backbone consisting of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues. Glycan chains are cross-linked by short peptides attached to MurNAc residues to form a mesh-like sacculus surrounding the cytoplasmic membrane. The importance of this macromolecule is highlighted by the number of antimicrobials that target PG and steps in its biosynthesis pathway.

Lysozyme is a muramidase of the innate immune system that hydrolyzes the β-1,4-glycosidic bond between MurNAc and GlcNAc residues, causing bacterial cell lysis. Because of the unique nature of PG, released fragments serve as important recognition motifs for immune receptors, activating the immune response in the early stages of an infection (5). Many pathogens have therefore evolved a strategy to defend against the host immune system through modification to their PG.

One such modification is O-acetylation of the C6-hydroxyl of MurNAc residues of PG, which sterically hinders binding of lysozyme (6, 7). This modification is widespread among Gram-negative and Gram-positive bacteria, but it is most predominant in pathogens (8–11). Bera et al. (12) discovered that only pathogenic species of Staphylococcus produce O-acetylated PG, and they are resistant to lysozyme. The levels of PG O-acetylation can range from 20 to 80% depending on the organism, environmental conditions, and growth phase of the culture (13–15). For example, the levels of PG O-acetylation increase by 10–40% as Enterococcus faecalis cells enter stationary the phase and a further 10–16% as the cells enter the viable but nonculturable state (16). In addition to providing resistance to lysozyme, PG O-acetylation has important implications in virulence, including increasing disease severity and downstream complications (17, 18), conferring resistance to bacteriocins (19) and β-lactam antibiotics (10), and influencing the immune response (19, 20). PG O-acetylation is considered important for virulence in numerous pathogens such as S. aureus (8, 17, 20), Streptococcus pneumoniae (10), Listeria monocytogenes (19), Neisseria meningitidis (21), Neisseria gonorrhoeae (9, 18), Helicobacter pylori (22), and E. faecalis (23).

In Gram-positive bacteria, the enzyme responsible for PG O-acetylation is O-acetyltransferase A (OatA), first identified in S. aureus (8). Homologs of OatA have since been identified in S. pneumoniae (10), L. monocytogenes (19), E. faecalis (23), Lactobacillus plantarum (24), Lactococcus lactis (25), and several other Staphylococcus species (12). OatA is a bimodular protein consisting of an N-terminal acyltransferase 3 integral membrane domain and an extracellular C-terminal SGNH/GDSL-hydrolase domain. SGNH hydrolases are a large family of esterases and lipases that possess four consensus residues, Ser, Gly, Asn, and His, that comprise their active sites and are involved in their mechanism of action (26). The catalytic Ser of these enzymes is found in a GDSL sequence motif. The N-terminal domain of OatA is predicted to contain 11 transmembrane helices and is thought to shuttle acetyl groups across the cytoplasmic membrane to the C-terminal domain for their subsequent transfer onto PG (27). It is still unknown whether the two domains remain attached after translation; S. aureus OatA possesses a noncanonical signal peptidase site between the two domains, and the C-terminal domain alone has been detected in spent culture media (28).

We recently described the crystal structure of the C-terminal domain of OatA from S. pneumoniae and experimentally confirmed the function of this domain as an O-acetyltransferase with a reaction mechanism involving a Ser-His-Asp catalytic triad (29, 30). Preliminary characterization of the C-terminal domain of S. aureus OatA was also performed, including identification of the putative catalytic triad residues. Furthermore, the substrate specificity of the C-terminal domains of S. pneumoniae and S. aureus OatA was investigated with regard to the stem peptide, and it was found that the enzymes had distinct preferences for muroglycans with tetra- and pentapeptide stems, respectively (29). Given that PG O-acetylation is a postbiosynthetic modification, occurring after incorporation of lipid II precursors into the pre-existing sacculus (31–34), OatA must work intimately with the PG biosynthetic machinery. Here, we present the crystal structure of the C-terminal domain of S. aureus OatA and characterization of its mechanism of action as an O-acetyltransferase. Examination of the active center suggests that the staphylococcal enzyme, as well as those produced by most other Gram-positive pathogens, use a novel process for preventing simple hydrolysis of the acetyl-enzyme intermediate compared with the previously characterized streptococcal OatA.

Results

Crystallization and structure determination of SaOatA_C

Efforts to crystallize the engineered extracytoplasmic domain of S. aureus OatA, encompassing residues 445–601 (SaOatA_C), were unsuccessful, despite the removal of potentially disordered regions that could hinder crystallization. This included significant truncation of the N-terminal interdomain-linker regions and two C-terminal lysine residues. To find additional areas of disorder, we analyzed the amino acid sequence of SaOatA_C and found that it was enriched with residues of high conformational freedom (9.6% Lys and 5.1% Glu). Therefore, we used surface entropy reduction (35) in an effort to reduce surface disorder and promote crystallization of the domain. We identified three clusters of predicted high entropy surface residues: Lys⁴⁶⁴ and Lys⁴⁶⁵, Lys⁴⁹⁵ and Lys⁴⁹⁶, and Glu⁵⁵¹ and Lys⁵⁵², using the SERp server. Each residue in these cluster pairs was replaced with alanine residues to produce three new constructs for crystallization. The specific activities of the resultant SaOatA_C variants were similar to WT enzyme (Table 1), and all three variants crystallized.

Table 1.

Specific activities of SaOatA_C variants

± denotes standard deviation; ND, no detectable activity.

Enzyme variant	Esterase activitya		Transferase activityb		Tmc
	Specific activity	Relative activity	Specific activity	Relative activity
	nmol min−1 mg−1	%	nmol min−1 mg−1	%	°C
SaOatAC (WT)	6.78 ± 0.13	100
K464A/K465A	7.18 ± 0.090	106
K495A/K496A	7.70 ± 0.042	114			57.35 ± 0.163
E551A/K552A	7.83 ± 0.057	115
SaOatAC (WT)	4.88 ± 0.15	100	0.49 ± 0.19	100	59.49 ± 0.025
S453A	ND	0			54.83 ± 0.233
H578A	0.120 ± 0.0034	2.08			56.96 ± 0.086
D575A	0.060 ± 0.0033	1.06			51.30 ± 0.234
N507A	ND	0			51.26 ± 0.053
V475G	ND	0			51.32 ± 0.057
D457A	18.5 ± 0.20	392	4.25 ± 0.73	866	57.77 ± 0.310
D457N	18.1 ± 0.18	385	3.25 ± 0.80	662	57.59 ± 0.376

^a The reactions were conducted in 50 mm sodium phosphate at pH 7.0 for the first four variants and pH 6.5 for all others at 25 °C with 0.1 mm 4MU-Ac.

^b The reactions were conducted in 50 mm sodium phosphate at pH 7.0 at 37 °C with 0.1 mm 4MU-Ac and 2 mm pentaacetyl-chitopentaose

^c The T_m values were determined for purified SaOatA_C using the thermal shift assay with SYPRO Orange.

(E551A/K552A)-SaOatA_C crystallized only in the presence of zinc salts, so we suspected that Zn²⁺ ions were bound to the protein and well-ordered. Indeed, we detected an anomalous signal from protein-bound zinc, and this signal was sufficient for phase determination using single-wavelength anomalous dispersion (Zn-SAD) (Table 2). (E551A/K552A)-SaOatA_C crystallized with a dimer in the asymmetric unit and contained three Zn²⁺ions, two of which are coordinated by the putative catalytic His and Asp residues at the dimer interface. We presume that these protein-bound Zn²⁺ ions are not biochemically relevant because they appear to distort the active site. This was supported by the observation that zinc, among other first-row transition metal cations, inhibits SaOatA catalysis in vitro (36). Diffraction data were therefore collected for a crystal of (K495A/K496A)-SaOatA_C that grew in the absence of zinc, and the structure was solved by molecular replacement using the (E551A/K552A)-SaOatA_C structure as a search model. This structure was determined to 1.71 Å resolution and refined to R_work/R_free values of 16.6%/19.6% (Table 2).

Table 2.

Summary of data collection and refinement statistics

	(K495A/K496A)-SaOatAC	(E551A/K552A)-SaOatAC
Data collection
Beamline	NSLS-II 17-ID2	CLS 08B1-1
Wavelength (Å)	0.99961	1.28167
Space group	P 21	P 21 21 21
Unit cell parameters
a (Å)	42.35	39.51
b (Å)	61.30	78.86
c (Å)	67.69	106.59
α (°)	90.00	90.00
γ (°)	90.00	90.00
β (°)	100.90	90.00
Resolution range (last shell) (Å)	28.78–1.71 (1.77–1.71)	44.16–1.55 (1.61–1.55)
Total no. reflections (last shell)	71,747 (6748)	574,457 (19,959)
No. unique reflections (last shell)	36,587 (3545)	46,179 (2983)
Redundancy (last shell)	1.96 (1.90)	12.43 (6.69
Completeness (last shell) (%)	99.36 (96.12)	93.85 (68.8)
Average I/σI (last shell)	17.32 (1.97)	17.53 (4.11)
Rmerge (last shell, %)a	0.02392 (0.4006)	0.09615 (0.3412)
CC½ (last shell)b	0.999 (0.633)	0.997 (0.97)
Refinement
Rwork/Rfreec	0.166/0.196	0.1720/0.1923
No. atoms	2693	2840
Protein	2423	2450
Water	268	389
Ligand	2	3
Average B-factor (Å)	31.84	24.82
Protein	30.74	23.41
Water	41.88	33.73
Ligand	26.85	18.46
Root mean square	0.007	0.005
Bond lengths (Å)
Bond angles (°)	0.82	0.73
Ramachandran total (%)	99.01	98.04
Favored
Allowed	0.99	1.96
PDB entry	6VJP	6WN9

^aR_merge = Σ_hklΣ_i|(I_i(hkl) − <I(hkl)>|/Σ_hklΣ_iI_i(hkl), where I is the intensity of the reflection hkl, Σ_hkl is the sum over all reflections, and Σ_i is the sum over i measurements of reflection hkl. <I(hkl)> is the mean value of I(hkl).

^b CC_½ is the Pearson correlation coefficient calculated between two random half data sets. CC = Σ(x − <x>)(y − <y>)/[Σ(x − <x>)²Σ(y − <y>)²]^½.

^c R_work = Σ_hkl|F_obs − F_calc|/Σ_hkl|F_obs|, where F_obs and F_calc are the observed and calculated structure factors, respectively. R_free was calculated identically except that all reflections belonged to a test set consisting of only 5% of the data, chosen at random.

The overall structure of (K495A/K496A)-SaOatA_C adopts an α/β-hydrolase fold with five parallel β-strands sandwiched between seven α-helices (Fig. 1). The asymmetric unit contains two molecules of the protein; however, size-exclusion chromatography revealed that SaOatA_C is monomeric, and thus the dimeric conformation seen in the crystal is likely not physiologically relevant. A sodium ion was seen in the bend of α-helix 4 and β-strand 5, coordinated by three water molecules and the backbone carbonyl oxygens of Ala⁵⁵⁰, Arg⁵⁵³, and Val⁵⁵⁶. The putative catalytic triad Ser-His-Asp residues align within a shallow active site on the surface of the protein. In chain B, the catalytic His⁵⁷⁸ is found in two conformations, facing both toward and away from the active site (occupancies of 0.54 and 0.46, respectively), suggesting that His⁵⁷⁸ is inherently flexible (Fig. S1).

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.

Searching for structural homologs with DALI revealed that SaOatA_C most closely resembles the C-terminal domain of OatA from S. pneumoniae (SpOatA_C) (PDB code 5UFY) (Fig. 2A). The SaOatA_C structure is also homologous to numerous uncharacterized proteins proposed to belong to the SGNH/GDSL hydrolase family. Of the characterized proteins in the PDB database, aside from SpOatA_C, SaOatA_C most closely resembles Axe2, an acetylxylan esterase from Geobacillus stearothermophilus (PDB code 4JHL), a family 3 carbohydrate esterase from Clostridium thermocellum (PDB code 2VPT), and a family 3 carbohydrate esterase from Talaromyces cellulolyticus (PDB code 5B5S) (Fig. 2A). The most prominent difference between SaOatA_C and its structural homologs is the geometry of the putative catalytic triad. The distances between Nδ1 of His⁵⁷⁸ and Oδ2 of Asp⁵⁷⁵ and between the ϵ2 of His⁵⁷⁸ and Oγ of Ser⁴⁵³ are 3.2 and 4.3 Å, respectively, which are significantly longer than in homologs (Fig. 2B). However, in the absence of a bound ligand, the crystal structure of SaOatA_C could represent the inactive resting state of the enzyme and not the active conformation. Indeed, the dual conformations of His⁵⁷⁸ seen in chain B of the crystal structure suggests that His⁵⁷⁸ is flexible (Fig. S1), and the active site may undergo conformational changes during catalysis. Such conformational changes have been reported previously for SpOatA_C and the PG esterase Ape1 (29, 37).

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.

SaOatA_C uses a Ser-His-Asp catalytic triad

SGNH hydrolases are characterized by four consensus sequence blocks (I, II, III, and V) containing conserved residues, Ser, Gly, Asn, and His that, as noted above, give this family of enzymes their name (Ser⁴⁵⁴, Gly⁴⁷⁵, Asn⁵⁰⁷, and His⁵⁷⁸ in SaOatA) (26). Most SGNH hydrolases possess a catalytic triad consisting of a Ser from block I and an Asp and His from block V. The putative catalytic triad residues of SaOatA_C, Ser⁴⁵³, His⁵⁷⁸, and Asp⁵⁷⁵ were previously replaced with Ala in a longer construct of the enzyme (SaOatA_435–603) and assayed for activity using p-nitrophenyl-acetate (pNP-Ac) (29). We repeated these experiments using the new construct with 4-methylumbelliferyl-acetate (4MU-Ac) as substrate. The stability of all SaOatA_C variants was verified by a thermal shift assay after purification. The (S453A)-SaOatA_C variant had no detectable activity, whereas the (H578A)-SaOatA_C and (D575A)-SaOatA_C variants had 2.08% and 1.02% residual esterase activity, respectively (Table 1). As seen previously, WT SaOatA_C displays very limited transferase activity toward chito-oligosaccharides as acceptors (29), and as such, it was not possible to determine the rates of transfer for all SaOatA_C variants. A qualitative analysis of any reaction products by LC-MS demonstrated that the truncated WT SaOatA_C retained transferase activity; however, the sensitivity of the assay is not sufficient to discriminate between no transfer and limited transfer that may be seen with weakly active SaOatA_C variants (Fig. S2).

Most SGNH hydrolases employ a double-displacement reaction mechanism involving a covalent acyl-enzyme intermediate at the catalytic serine residue. Indeed, this reaction mechanism was recently confirmed for SpOatA_C (30). We employed a similar strategy to unambiguously assign the role of the putative catalytic Ser⁴⁵³. We observed an accumulation of acetyl-SaOatA_C intermediate by real-time analysis of a reaction with pNP-Ac as acetyl donor using LC-MS (Fig. 3A). SaOatA_C had a molecular mass of 17,562 Da. After incubation with pNP-Ac for 15 min, we observed the appearance of multiply charged ions with a decovoluted molecular mass of 17,604 Da. The difference of 42 Da is consistent with the addition of an acetyl group. To identify the acetylated amino acid, we quenched an identical reaction mixture with acetone after 30 min of incubation and digested the recovered enzyme with trypsin. The resulting peptides were separated and analyzed by LC-MS/MS. We observed a pair of peptides, m/z 1117.59 and 1138.59, with an m/z difference of 42. The mass and fragmentation patterns of these parent ions correspond well with the native and acetylated forms, respectively, of the peptide ⁴⁴²AASSPLIGDSVMVDIGNVFTK⁴⁶⁴ (with Ser⁴⁵³ in bold) (Fig. 3B). Based on the MS/MS fragmentation pattern of the m/z 1138.59 parent ion, we were able to map the acetyl modification to Ser⁴⁵³. This m/z 1138.59 ion was not detected among the digestion products of the no-substrate control reaction.

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.

SaOatA_C possesses a typical three-component oxyanion hole

In most SGNH hydrolases, the oxyanion hole is typically formed by three conserved hydrogen-bond donors: the backbone amide of the catalytic Ser of the block I consensus sequence, the backbone amide of Gly from block II, and the side-chain amide of Asn from block III. In SpOatA_C, the block II Gly is replaced by a Ser, and the loop adopts a type I β-turn. In contrast, SaOatA_C retains the Gly, and the loop adopts the typical type II β-turn seen in homologous SGNH esterases (Fig. 2B). The backbone amide of Gly⁴⁷⁶ in SaOatA_C faces the active site and thus likely participates in stabilizing the transition state. Interestingly, the Nδ² of block III Asn⁵⁰⁷ is only 3.1 Å away from the Oγ of Ser⁴⁵³, closer than typically seen in other SGNH hydrolases. We tested the importance of Asn⁵⁰⁷ in catalysis by its replacement with Ala. The (N507A)-SaOatA_C variant had no detectable esterase activity toward 4MU-Ac, suggesting an important role in the catalytic mechanism (Table 1).

We also previously proposed the importance of a (V/I)(G/S)(R/V) motif in the block II loop (29). In the resting-state structure of SpOatA_C, a water molecule was observed coordinated by the backbone carbonyl of Val⁴⁶⁰ and the backbone carbonyl of Val⁴⁶² (equivalent residues Val⁴⁷⁵ and Arg⁴⁷⁷ in SaOatA_C) (29). In contrast, because of the opposite turn of the block II loop in SaOatA_C, the carbonyl of Val⁴⁷⁵ does not face the active site, and no water molecule is seen coordinated at the active site in this position (Fig. 4 and Fig. S3). Nonetheless, Val⁴⁷⁵ is highly conserved among OatA homologs, and in accordance, we replaced Val⁴⁷⁵ with Gly and saw complete abolishment of esterase activity (Table 1).

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.

Conserved Asp⁴⁵⁷ limits esterase activity

We observed a water molecule in the active site of SaOatA_C coordinated by the Oδ1 of Asp⁴⁵⁷, the backbone carbonyl of Ile⁵⁷⁷, and the Oγ of the catalytic Ser⁴⁵³ that was not present in any of the structural esterase homologs or SpOatA_C (Fig. 4). We investigated the role of the water molecule by substituting Asp⁴⁵⁷ with Ala and Asn. Surprisingly, the D457A and D457N variants of SaOatA_C displayed a 4-fold increase in esterase activity and still maintained the ability to transfer to chito-oligosaccharides (Table 1). To investigate the prevalence of an Asp at this position in SGNH hydrolases/transferases, we analyzed sequences from 200 known and hypothetical OatA homologs from Gram-positive bacteria and characterized esterases from the SGNH hydrolase family. We found that an Asp is highly conserved in OatA homologs from the Staphyloccocus, Bacillus, Listeria, and Lactobacillus genera, among others (Fig. S4). In contrast, an Asp residue was not found at this position (catalytic Ser + 4) in any of the characterized SGNH hydrolases that naturally act as esterases (Fig. 2C). In most OatA homologs from Streptococcus species, an Arg residue is found in this position (Fig. S4). We propose that Asp⁴⁵⁷ is a conserved feature in many SGNH family transferases and serves as a sentry to limit esterase activity through the coordination of a water molecule that could otherwise approach an acetyl-enzyme intermediate. It was observed that transferase activity also increased with the replacement of Asp⁴⁵⁷ with Ala or Asn. The reason for this enhanced activity is not known, but it is possible that these replacements increase the accessibility of the pseudo-substrate chitooligosaccharides, in addition to water, used as acceptors for the in vitro assays of transferase activity.

Discussion

OatA belongs to the SGNH hydrolase family of enzymes along with numerous esterases with a wide range of substrate specificities. The mechanism by which OatA acts as a transferase was widely unknown until recently. The structure of SpOatA_C elucidated structural features that distinguished it from SGNH hydrolase family esterases, including an inverted turn of the block II loop, a conserved valine in block II, a hydrophobic active site wall, and an atypical two-residue oxyanion hole (29). Unexpectedly, the active site of SaOatA_C more closely resembles that of structurally homologous esterases than that of SpOatA_C. SaOatA_C has the conserved Gly in block II, and the loop adopts the typical type II β-turn seen in the homologous esterases. As a consequence, the water molecule that is coordinated by the backbone of Val⁴⁶⁰ in the block II sequence of SpOatA_C in its resting state is not seen in the structure of SaOatA_C. Furthermore, replacement of Val⁴⁶⁰ of SpOatA_C with Gly or Ala increases esterase activity while resulting in loss of transferase activity (29). To explain this loss, it was proposed that Val⁴⁶⁰ may contribute to the effective binding of the carbohydrate acceptor. In contrast, a comparative replacement of the homologous Val in SaOatA_C, Val⁴⁷⁵ resulted in a total loss of esterase activity. It is possible that replacement of Val⁴⁷⁵ with Gly in SaOatA_C disrupts correct positioning of the block II loop, which may impact the ability of the backbone amide of Gly⁴⁷⁶ to stabilize the transition state; such stabilization of the transition state formed by SpOatA_C does not appear to involve its block II loop (29).

A thorough kinetic analysis of SpOatA_C confirmed that the enzyme employs a double-displacement reaction mechanism (30). Accordingly, we propose that SaOatA_C follows a similar reaction mechanism (Fig. S5), wherein the carboxyl group of Asp⁵⁷⁵ forms a salt bridge with a nitrogen atom in the imidazole ring of His⁵⁷⁸, enabling His⁵⁷⁸ to deprotonate Ser⁴⁵³. The nucleophilic Ser⁴⁵³ attacks the carbonyl carbon of the acetyl donor, generating a tetrahedral transition state. Residues at the active center of the enzyme form an oxyanion hole that stabilizes the transition state, which then collapses into a covalently bound acetyl-enzyme intermediate. We were able to unequivocally identify Ser⁴⁵³ of SaOatA_C as the site of acetylation (Fig. 3). The acetyl donor is released upon acquisition of a proton from His⁵⁷⁸. The glycan accepter, a MurNAc residue of the PG backbone, can then bind the active site cleft. His⁵⁷⁸, now acting as a base, abstracts a proton from the C6-OH of MurNAc, rendering the carbon atom nucleophilic and resulting in its attack on the carbonyl center of the acetyl-Ser⁴⁵³ intermediate. This leads to the formation of a second tetrahedral transition state, collapse of which results in the release of the O-acetylated product and free enzyme.

We propose that the transition state is stabilized by the backbone amide of Ser⁴⁵³ in block I, the side-chain amide of Asn⁵⁰⁷ in block III, and the backbone amide of Gly⁴⁷⁶ in block II. The three-residue oxyanion hole is typical of SGNH hydrolases, but distinguishes SaOatA_C from SpOatA_C, which appears to employ an oxyanion hole formed of two residues (29). Replacement of the SpOatA_C block III Asn⁴⁹¹ with Ala gave 58% residual esterase activity (29). Further kinetic analysis suggested that Asn⁴⁹¹ may play a larger role in substrate binding than in stabilization of the first transition state (30). In contrast, replacement of Asn⁵⁰⁷ of SaOatA_C with Ala resulted in a complete loss of activity, suggesting that this residue may play a more critical role in SaOatA_C. Absolute identification of oxyanion hole H-donors would require analysis of a ligand-bound structure, ideally with a covalently bound transition-state mimic. Such a structure was achieved for SpOatA_C using the mechanistic inhibitor methanesulfonyl fluoride, forming a methylsulfonyl-adduct structure (PDB code 5UG1). Unfortunately, methanesulfonyl fluoride and related analogs do not significantly inhibit SaOatA_C; thus a different transition state mimic will need to be found.

Bioinformatic analysis shows that OatA homologs form two distinct clades, wherein the Streptococcus genus forms a phylogenetically separate clade to Staphylococcus, Bacillus, and other genera (Fig. S4). The differences that we have observed between the structures of S. pneumoniae and S. aureus OatA suggest that the enzymes from these clades may use different mechanisms to minimize, if not prevent, water from serving as the acetyl acceptor during their respective double-displacement reaction mechanisms. We previously noted the occurrence of a conserved Val/Ile adjacent to the oxyanion hole block III Asn in Streptococcus OatA homologs, proposing that this hydrophobic residue may stabilize carbohydrate acceptor substrates (29). In contrast, in Staphylococcus and species from the same clade, this position is most commonly occupied by a Thr or Ser residue, which would not engage in the same hydrophobic interactions. We identified a water molecule coordinated by Asp⁴⁵⁷ in the structure of SaOatA_C and determined that this residue played an important role in limiting the esterase activity of the enzyme, while maintaining transferase activity. This suggests that the two distinct clades of OatA homologs utilize different mechanisms to preclude water from their active site to catalyze efficient and nonwasteful transfer of acetyl groups to peptidoglycan only. Our data suggest that the coordination of a water molecule in the active site by Asp⁴⁵⁷ may be the method by which SaOatA_C and the majority of OatA homologs belonging to the same phylogenetic clade favor transferase activity. Unfortunately, the absence of other OatA structures prevents us from verifying whether or not these structural features are conserved among homologs within the same clade. The reason for these differences also remains unknown. Perhaps the selective pressure for divergence into two clades was substrate specificity recognizing that the staphylococcal OatA O-acetylates MurNAc residues with pentapeptide stems, whereas the streptococcal enzyme has specificity for residues with tetrapeptide stems (29).

PG O-acetylation is a common modification employed by pathogenic Gram-positive bacteria as a means to evade the host innate immune system. Despite knowledge of the modification for decades, OatA from S. pneumoniae was the only PG O-acetylating enzyme from a Gram-positive bacteria characterized before this study. Our data reinforce the mechanism of action proposed for both S. pneumoniae OatA_C and N. gonorrhoeae peptidoglycan O-acetyltransferase B (PatB) (30, 38). We have previously validated SaOatA_C and N. gonorrhoeae PatB as antibiotic targets with a high-throughput small-molecule screen (39). The structure of SaOatA_C will assist in the design of anti-virulence drugs against OatA. Furthermore, our discovery of the differences between the active sites of S. pneumoniae and S. aureus OatA_C is an important consideration in developing narrow- or broad-spectrum OatA inhibitors for the treatment of important human pathogens for which current antibacterial therapies are being threatened by multidrug resistance.

Experimental procedures

Cloning of C-terminal S. aureus oatA variants

The generation of SaOatA_C(445–601) possessing single site-specific amino acid replacements was achieved by site-directed mutagenesis. PCR products incorporating the desired mutations were obtained using KAPA HiFi polymerase with pDSAC71 (harboring oatA_C encoding residues 445–601 of full-length S. aureus OatA) as template and the appropriate primers listed in Table S1. Following PCR amplification, the reaction was incubated with Dpn1 (Thermo Fisher Scientific, Mississauga, Canada) for 1 h at 37 °C, followed by transformation into Escherichia coli DH5α. The sequences of all resultant plasmids were verified before use.

Overproduction and purification of SaOatA_C

The genes encoding SaOatA_C(445–601) and variants were expressed in E. coli BL21 (DE3) transformed with pDSAC71, and the overproduced recombinant proteins were purified by a combination of affinity chromatography and gel filtration as previously described (39). Gel filtration buffer consisting of 50 mm Tris, pH 7.5, 150 mm NaCl was used when the protein was being purified for the purpose of crystallography. In all other instances, the gel filtration buffer consisted of 50 mm sodium phosphate, pH 6.5, 150 mm NaCl. Fresh immobilized metal affinity chromatography resin was used for each SaOatA_C variant to prevent cross-contamination.

The stability of each purified protein was assessed by SYPRO Orange thermal shift assay as a means to infer proper folding (40). Briefly, SaOatA_C and variants were diluted to 5 μm in 50 mm sodium phosphate, pH 7.0, and mixed with 2× SYPRO Orange (Thermo Fisher Scientific) in 50-μl reactions. The melting temperature (T_m) of each protein was determined using a StepOnePlus real-time PCR machine using a temperature gradient of 4 to 95 °C over 60 min. The data were analyzed using the StepOnePlus software.

Crystallization

SaOatA_C surface entropy variants were concentrated to 30 mg/ml by ultrafiltration using an Amicon Ultra-15 centrifugal filter (10-kDa molecular mass cutoff; Millipore) (4,000 × g, 4 °C). Commercial Midwest Center for Structural Genomics crystallization suite sparse matrix crystallization screens 1–4 (Microlytic North America Inc., Burlington, MA, USA) were prepared at room temperature with the E551A/K552A and K495A/K496A forms of SaOatA_C. Crystallization screening by sitting-drop vapor diffusion was set up using a Gryphon robot (Art Robbins Instruments, Sunnyvale, CA, USA) with 1-μl protein drops and a protein-to-reservoir ratio of 1:1 for a final drop volume of 2 μl. Crystal trays were stored at 22 °C. Optimization of crystal conditions was performed to produce crystals of (E551A/K552A)-SaOatA_C in 0.008 m zinc acetate, 20% PEG 3350, and crystals of (K495A/K496A)-SaOatA_C in 27% PEG 6000, 0.015 m sodium citrate.

X-ray diffraction data collection and structure determination

Crystals were cryoprotected for 30 s in reservoir solution supplemented with 60% (v/v) ethylene glycol prior to vitrification in liquid nitrogen. Zn-SAD data for (E551A/K552A)-SaOatA_C were collected on Beamline 08B1-1 at the Canadian Synchrotron Light Source (Saskatoon, Canada). Native data for (K495A/K496A)-SaOatA_C were collected on Beamline 17-ID2 at the National Synchrotron Light Source II (Upton, NY). The data were indexed and scaled using HKL2000 (41). Three zinc sites were located in the (E551A/K552A)-SaOatA_C Zn-SAD data using HKL2MAP (42), and density modified phases were calculated using SOLVE/RESOLVE (43). The resulting electron density map was of good quality and allowed for PHENIX AutoBuild (44) to build 100% of the protein. Manual model building was done in COOT (45) alternated with refinement using PHENIX.REFINE (46). The structure of native (K495A/K496A)-SaOatA_C was determined by molecular replacement using PHENIX AutoMR (46) with the zinc-incorporated derivative as the search model. Manual model building and refinement was carried out as described previously. All molecular models were generated using PyMOL.

Steady-state kinetics of SaOatA_C

The specific activity of SaOatA_C acting as an esterase and transferase was determined as previously described (39). Briefly, SaOatA_C (5 μm) was incubated in 50 mm sodium phosphate, pH 6.5, at room temperature with 0.1 mm 4MU-Ac as substrate. For transferase assays, SaOatA_C (5 μm) was incubated in 50 mm sodium phosphate, pH 6.5, at 37 °C with 0.1 mm 4MU-Ac as acetyl donor and 2 mm pentaacetyl-chitopentaose (Megazyme) as acetyl acceptor. Product release was monitored fluorometrically using a Synergy plate reader with excitation and emission wavelengths of 325 and 450 nm, respectively. Control reactions were performed using gel-filtration buffer in place of SaOatA_C to account for spontaneous substrate hydrolysis. The rate of background hydrolysis was subtracted from the rate of reactions with enzyme to determine a rate of esterase activity. The rate of transfer was determined as the difference between the reaction rates with and without acceptor. Each reaction was performed in triplicate. Analyses and graphs were generated in GraphPad Prism 5.

Qualitative analysis of SaOatA_C transferase activity

The ability of SaOatA_C to transfer acetyl groups to chito-oligosaccharides was determined by qualitative end-point analysis by LC-MS/MS. SaOatA_C (5 μm) was incubated with 0.1 mm 4MU-Ac and 1 mm pentaacetyl-chitopentaose (Megazyme) at 37 °C for 18 h. The reaction products were separated by LC-MS/MS using an Agilent 1200 HPLC system interfaced with an Agilent UHD 6520 Q-TOF mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) housed in the Mass Spectrometry Facility of the Advanced Analysis Centre of the University of Guelph. Data analysis was performed using Mass-Hunter qualitative analysis, version B.06.00 (Agilent).

Trapping acetyl-SaOatA_C intermediate

Direct observation of the covalent acetyl-SaOatA_C intermediate was achieved by real-time analysis of a reaction by LC-MS as previously described (30) with minor modifications. The reaction mixture consisted of 5 μm SaOatA_C in 50 mm sodium phosphate, pH 6.5, with 1 mm pNP-Ac as acetyl donor and was incubated for 15 min in an MS-grade sample vial. A control reaction without pNP-Ac was also performed. The site of O-acetylation was identified by a tryptic digest of an acetone-quenched reaction mixture of SaOatA_C with pNP-Ac, as previously described (30). In this case, the reaction mixture consisted of 5 μm SaOatA_C in 50 mm sodium phosphate, pH 6.5, with 1 mm pNP-Ac, incubated for 30 min in an MS-grade sample vial. A control reaction without pNP-Ac was also performed. All samples and experiments were run as previously described (30). Data analyses were performed using MassHunter qualitative analysis, version B.06.00 (Agilent).

Other analytical procedures

Nucleotide sequencing of PCR products and plasmids was performed by the Genomics Facility of the Advanced Analysis Center (University of Guelph). Protein concentrations were determined using the Pierce BCA protein assay kit with BSA serving as the standard. SDS-PAGE on 15% acrylamide gels was conducted by the method of Laemmli (47) with Coomassie Brilliant Blue staining. Surface entropy reduction analyses were conducted using the SERp server (35).

Data availability

The atomic coordinates and structure factors of the reported crystal structures have been deposited in the Protein Data Bank under codes 6VJP and 6WN9. The authors declare that all other data supporting the findings of this study are available within the paper and its supporting information.