Substrate specificity and kinetic characterization of peptidoglycan O-acetyltransferase B from Neisseria gonorrhoeae

Article abstract of DOI:10.1074/jbc.M114.567388

The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neisseria meningitidis. With Gram-negative bacteria, the translocation of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidoglycan by O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics.

Full text of DOI:10.1074/jbc.M114.567388

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 24, pp. 16748–16760, June 13, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Substrate Specificity and Kinetic Characterization
of Peptidoglycan O-Acetyltransferase B from
Neisseria gonorrhoeae*
Received for publication, March 25, 2014, and in revised form, April 17, 2014 Published, JBC Papers in Press, May 2, 2014, DOI 10.1074/jbc.M114.567388
Patrick J. Moynihan and Anthony J. Clarke1
From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Background: The O-acetylation of peptidoglycan in Gram-negative bacteria is catalyzed by PatB.
Results: PatB can utilize a variety of acetyl donors for transfer to polymerized glycans containing N-acetyl groups and muro-
glycans containing tri- and tetrapeptide stems.
Conclusion: PatB has broad specificity for acetyl donors but narrow specificity for acceptor glycans.
Significance: This information will help with the identification of possible leads to novel classes of antibiotics.
The O-acetylation of the essential cell wall polymer pepti-
doglycan is a major virulence factor identified in many bacteria,
both Gram-positive and Gram-negative, including Staphylococ-
cus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neis-
seria meningitidis. With Gram-negative bacteria, the transloca-
tion of acetyl groups from the cytoplasm is performed by an
integral membrane protein, PatA, for its transfer to peptidogly-
can by O-acetyltransferase PatB, whereas a single bimodal mem-
brane protein, OatA, appears to catalyze both reactions of the
process in Gram-positive bacteria. Only phenotypic evidence
existed in support of these pathways because no in vitro bio-
chemical assay was available for their analysis, which reflected
the complexities of investigating integral membrane proteins
that act on a totally insoluble and heterogeneous substrate, such
as peptidoglycan. In this study, we present the first biochemical
and kinetic analysis of a peptidoglycan O-acetyltransferase
using PatB from N. gonorrhoeae as the model system. The
enzyme has specificity for muropeptides that possess tri- and
tetrapeptide stems on muramyl residues. With chitooligosac-
charides as substrates, rates of reaction increase with increasing
degrees of polymerization to 5/6. This information will be valu-
able for the identification and development of peptidoglycan
O-acetyltransferase inhibitors that could represent potential
leads to novel classes of antibiotics.
␤-lactams and other clinically introduced antibiotics that fol-
lowed has necessitated the search for new antibacterial targets.
One such target may be the enzymes involved in the metabo-
lism of PG O-acetylation.
The O-acetylation of PG is a major virulence factor identified
in many bacteria, both Gram-positive and Gram-negative. The
list includes important human pathogens, such as Staphylococ-
cus aureus, Bacillus anthracis, all species of Enterococcus, spe-
cies of Campylobacter, Listeria monocytogenes, Neisseria gon-
orrhoeae, and Neisseria meningitidis (reviewed in Ref. 1). This
modification to PG occurs predominantly at the C-6 hydroxyl
group of N-acetylmuramyl (MurNAc) residues, and it is partic-
ularly significant because it sterically hinders the activity of
muramidases (lysozymes), thereby providing protection from
the defensive activity of innate immune systems. Moreover, it
totally precludes the function of the lytic transglycosylases,
endogenous bacterial enzymes essential for cell growth and
division, PG turnover, and the insertion of wall-spanning struc-
tures, such as flagella and secretory systems (reviewed in Refs. 1
and 2). The lytic transglycosylases are also muralytic enzymes
with the same specificity as lysozyme, but they are not hydro-
lases. Rather, they perform substrate-assisted catalysis, utilizing
the C-6 hydroxyl group of MurNAc residues to cleave PG with
the concomitant formation of 1,6-anhydromuramic acid reac-
tion products (3). Consequently, the O-acetylation of PG has
been proposed to control the autolytic activity of the lytic trans-
glycosylases at the substrate level (1, 2). This PG modification
may also serve as a defense mechanism against lytic enzymes
produced by bacteriophage or Type VI secretion effectors,
although this remains to be investigated.
Bacterial cell walls and, in particular, their essential compo-
nent peptidoglycan (PG)2 have been a popular and successful
target for antibacterial therapy ever since penicillin was intro-
duced to clinicians in the 1940s. However, resistance to the
* This work was supported by Canadian Institutes of Health Research Team
Grant TGC114045 (to A. J. C.) and an Ontario Graduate Scholarship from
the Province of Ontario (to P. J. M.).
Recent reports have demonstrated that the C-6 hydroxyl of
N-acetylglucosaminyl (GlcNAc) residues in the PG of Lactoba-
cillus species also are acetylated (4), and this may extend to
species of Bacillus (5). With Lactobacillus plantarum, the
O-acetylation of GlcNAc appears to control its major autolysin
Acm2, an N-acetyl-glucosaminidase (which has specificity
for the GlcNAc-MurNAc glycosidic linkage instead of the
lysozyme-specific linkage of MurNAc-GlcNAc) (1, 4).
1 To whom correspondence should be addressed. Tel.: 519-824-4120; Fax:
519-837-1802; E-mail: a.clarke@exec.uoguelph.ca.
2 Theabbreviationsusedare:PG, peptidoglycan;MurNAc, N-acetylmuramicacid;
GlcNAc, N-acetylglucosamine;Ni2ϩ-NTA, Ni2ϩ-nitrilotriaceticacid;SPEGC, solid
phase extraction on graphitized carbon; ACN, acetonitrile; pNP-Ac, p-nitro-
phenol acetate; MU-Ac, 4-methylumbelliferyl acetate; ␣N-Ac, ␣-naphthyl ace-
tate; DP, degree of polymerization; GM2, GlcNAc-MurNAc-dipeptide; GM3,
GlcNAc-MurNAc-tripeptide;GM4, GlcNAc-MurNAc-tetrapeptide;SUMO, small
ubiquitin-like modifier; ESI, electrospray ionization; PBP, penicillin-binding
protein.
The O-acetylation of PG was discovered over 50 years ago by
two independent groups (6, 7), but the processes for this mod-
16748 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of Peptidoglycan O-Acetyltransferase B
EXPERIMENTAL PROCEDURES
ification were only recently described. Based on genomic stud-
ies, two distinct pathways were proposed that appeared to be
unique to Gram-positive and Gram-negative bacteria, respec-
tively (8), and subsequent experimental evidence has been
obtained to support both. Thus, a two-component system is
proposed for Gram-negative bacteria involving the transloca-
tion of acetyl groups from the cytoplasm (acetyl-CoA is the
presumed source) to the periplasm by an integral membrane
protein, PatA (PG O-acetyltransferase A) and then their
transfer to PG by a peripheral membrane O-acetyltransferase,
PatB (9, 10). With Gram-positive bacteria, a single bimodal
Chemicals and Reagents—Acrylamide and glycerol were
purchased from Fisher, whereas isopropyl ␤-D-1-thiogalacto-
pyranoside was from Roche Applied Science, and chitooligo-
saccharides were products of Toronto Research Chemicals or
Carbosynth (Berkshire, UK). All growth media were from
Difco. Ni2ϩ-nitrilotriacetic acid (Ni2ϩ-NTA)-agarose was sup-
plied by Qiagen (Valencia, CA), Source Q was purchased from
GE Healthcare, graphitized carbon solid phase extraction col-
umns (Carbograph SPE) were products of Grace Canada, Inc.
(Ajax, Canada), and Hypercarb porous graphitized carbon col-
membrane protein, Oat (O-acetylpeptidoglycan transferase), umns were supplied by Thermo Electron Corp. (Rockford, IL).
Mouse anti-His6 antibody was obtained from Santa Cruz Bio-
technology, Inc. Unless otherwise stated, all other chemicals
and reagents were purchased from Sigma-Aldrich.
appears to catalyze both reactions of the process (11, 12). Inter-
estingly, B. anthracis and strains of Bacillus cereus encode the
genes for both systems and in multiple copies (5). To date, how-
ever, only phenotypic evidence exists in support of these path-
ways in any bacterium because no in vitro biochemical assay
was available for their analysis. Moreover, none of these
enzymes have been demonstrated to modify their native sub-
strate in vitro. This situation reflects the complexities of inves-
tigating membrane-associated proteins that act on a totally
insoluble and heterogeneous substrate such as PG.
Isolation of Soluble Muropeptides—PG from Escherichia coli
was purified as described previously (1). An evenly dispersed 1.0
mg⅐mlϪ1 suspension of this PG (generated by sonication) in 50
mM sodium phosphate buffer, pH 6.2, containing 0.2% NaN3
and 5 mM MgCl2 was incubated at 37 °C for 24 h with both hen
egg white lysozyme and E. coli PBPs 4 and 7 (kindly provided by
K. Young, University of Arkansas School of Medicine) to a final
Ϫ1
concentration of 100 ␮g⅐ml and 10 ␮g⅐mlϪ1, respectively
Based on sequence alignment studies, the patB gene of
N. gonorrhoeae was originally predicted to encode an SGNH/
GDSL family esterase with similarity to the CAZy family CE-3
O-acetylxylan esterases (13) and Ape1 (O-acetyl-PG esterase 1)
(14), the latter being shown experimentally to function as a
serine esterase (15). Indeed, PatB shares 20.6% amino acid iden-
tity and 58.5% similarity with Ape1, including consensus motifs
involving a potential catalytic triad of residues expected of a
serine esterase. Whereas PatB was found to possess weak ester-
ase activity, phenotypic assays demonstrated its true function
to be a PG O-acetyltransferase (9). However, the lack of an
appropriate in vitro assay has precluded any understanding of
its substrate specificity and biochemical properties. To com-
pound this, the tertiary structure of any PG O-acetyltransferase
is not known. Consequently, lacking both a biochemical assay
and structural information, still nothing was known about the
enzymes despite increased interest in them. In this regard, an
inhibitor of Ape1 was demonstrated recently to serve as an
antibacterial with specificity against both Gram-positive and
Gram-negative bacteria that produce O-acetylated PG (16),
demonstrating the sensitivity of those bacteria to its alteration.
Furthermore, OatA from species of Staphylococcus has been
correlated with pathogenicity, whereas it is required for full
virulence in Listeria monocytogenes (17, 18). Given this,
together with the role PG O-acetylation has in controlling the
autolytic activity of the lytic transglycosylases (2, 9), it would be
expected that blocking this reaction through inhibition of PatB
would be even more deleterious to bacterial cells.
(PBPs 4 and 7 were included to enhance the production of sol-
uble, uncross-linked muropeptides). Insoluble material was
collected by centrifugation at 20,000 ϫ g, and the solubilized
muropeptides present in the supernatant were fractionated by
solid phase extraction as described below. The eluted muropep-
tides were dried in vacuo at 30 °C and stored at Ϫ20 °C prior to
use. The muramic acid content of respective samples was
determined by high pH anion exchange chromatography as
described previously (9).
Bacterial Strains and Culture Conditions—The strains of
bacteria used in this study and their genotypes are presented in
Table 1. N. gonorrhoeae FA1090 was grown for 24 h at 35 °C on
GC medium base supplemented with Kellogg’s defined supple-
ment (20, 21) in a humid, 5% CO2 environment, as described
previously (9). All plasmids constructed were screened and
maintained in E. coli DH5␣. When production of high levels of
protein was required, E. coli BL21*-␭DE3 cells were always
freshly transformed with the desired expression plasmid (Table
1) and grown in Super Broth (5 g of sodium chloride, 20 g of
yeast extract, and 32 g of tryptone) at 37 °C with agitation.
E. coli growth cultures were supplemented with chloramphen-
icol (35 mg⅐mlϪ1), ampicillin (100 mg⅐mlϪ1), and kanamycin
(50 mg⅐mlϪ1) when required.
Engineering of PatB—Due to low yields and protein instabil-
ity obtained with prior constructs (9), two new forms of PatB
were generated. The first involved a further truncation of the
enzyme by another 41 N-terminal amino acid residues. Thus,
the gene encoding PatB⌬36 was subcloned from pACPM22 (9)
by PCR amplification using the primers 3Ј-ATCGCTCGAGC-
TGTCCGGCGAAACGC-5Ј (forward) and 3Ј-CAGTAAGC-
TTTCATGGCTGTGTACTTGAGGTTG-5Ј (reverse). The
resulting PCR product was then digested using appropriate
restriction enzymes and ligated into a similarly digested
pBADHis-A plasmid with T4 DNA ligase, yielding pACPM30.
We recently reported the first in vitro assay for PG O-acetyl-
transferases that exploits their ability to utilize both chromo-
genic acetylesterase substrates as acetyl donors and chitooligo-
saccharides as acceptors (19). In this study, we present the first
biochemical analysis of a PG O-acetyltransferase using PatB
from N. gonorrhoeae as the model.
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Characterization of Peptidoglycan O-Acetyltransferase B
TABLE 1
Strains and plasmids used in this study
Strain/Plasmid
Description
Source/Reference
Strains
E. coli BL21*␭ (DE3)
E. coli DH5␣
FϪ ompT hsdSB (rBϪmBϪ) gal dcm rne131 (DE3)
Novagen
fhuA2 lac(del)U169 phoA glnV44 ⌽80Ј lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17
mcrA, ⌬(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ⌬lacX74, deoR, recA1, araD139,
⌬(ara-leu)7697, galU, galK, rpsL(SmR), endA1, nupG
Invitrogen
Invitrogen
E. coli Top10
Plasmids
pACPM22
pBADHis-A derivative encoding PatB truncated by its N-terminal 36 amino acid
residues with a C-terminal His6 tag; CmR
Ref. 9
pACPM30
pACPM33
pBAD His-A derivative encoding PatB truncated by its N-terminal
77 amino acid residues; AmpR
This study
This study
pET-SUMO derivative encoding PatB truncated by its N-terminal
36 amino acid residues with an N-terminal His6-SUMO tag; AmpR
The protein product of this construct, PatB⌬77, lacked its 77 (in triplicate) with p-nitrophenyl acetate (pNP-Ac) and chito-
N-terminal amino acids.
triose as substrates (19) in the following buffers: 50 mM ammo-
The second construct, encoding PatBSUMO, was also gener- nium acetate (pH 4 and 5), 50 mM sodium phosphate (pH 6–8),
ated by subcloning from pACPM22. In this case, PCR amplifi- and 50 mM Tris⅐HCl (pH 8.5 and 9). Activity was calculated
cation involved the forward primer 3Ј-TACTGGCAGCAGA- based on the total consumption of chitotriose as measured by
CCTACCAC-5Ј and the same reverse primer as described graphitized carbon HPLC (19).
above and ligation directly into the Champion pET-SUMO vec-
Q-TOF MS was used to analyze the initial transacetylation
tor according to the manufacturer’s specifications, yielding reaction products generated by PatBSUMO. Enzyme (1 ␮M) in 50
pACPM33. The protein product from this construct lacked its mM sodium phosphate buffer, pH 6.5, was incubated with 1 mM
36-amino acid, periplasmic-localizing signal sequence while chitopentaose and 4 mM pNP-Ac for 1 min, and then 10 ␮l of
possessing an N-terminal fusion of the His6-SUMO.
this reaction mixture was infused directly into the mass
Production and Purification of PatB—E. coli BL21*-␭DE3 spectrometer.
was transformed with pACPM30 or pACPM33 for overproduc-
The requirement of various metals for PatBSUMO activity was
tion of the recombinant forms of PatB described in this study. assessed by performing the assay as described above with the
Cells were grown in 1 liter of Super Broth at 37 °C to an A600 nm inclusion of 5 mM CaCl2, MgCl2, FeSO4, CuCl2, ZnSO4, or
of 0.6, and expression of genes was induced with isopropyl ␤-D- MnSO4. The effect of the metal chelator EDTA was also tested
1-thiogalactopyranoside (1 mM, final concentration) for 3 h. at a concentration of 5 mM. Other compounds assayed for their
Cells were harvested by centrifugation (6,000 ϫ g, 15 min, 4 °C), impact on PatBSUMO activity included NaCl (100 mM) and BSA
and they were resuspended in 30 ml of 20 mM sodium phos- (5 mg⅐mlϪ1).
phatebuffer, pH8.0, containing500mM NaCland10mM imida-
Determination of PatBSUMO Activity on Soluble Muro-
zole (lysis buffer) and subjected to lysis by four successive pas- peptides—To assess the ability of PatB to catalyze transfer of
sages through an EmusliFlex C-3 homogenizer (AVESTIN, acetyl groups to soluble PG fragments, microplate assays were
Inc., Ottawa, Canada) at 15,000 p.s.i. The lysate was clarified by performed as described above with soluble muropeptides
centrifugation (6,000 ϫ g, 15 min, 4 °C), and the PatB forms replacing chitotriose at a final concentration of 2 mM (based on
were isolated by affinity chromatography on Ni2ϩ-NTA-aga- muramic acid content). Completed reactions were pooled, and
rose. The resin (500 ␮l) was washed with the lysis buffer, and muropeptides were isolated from the reaction mixtures by solid
bound proteins were eluted in lysis buffer containing 250 mM phase extraction (described below). Eluted reaction products
imidazole. Recovered proteins were dialyzed at 4 °C exhaus- were analyzed by either MALDI-TOF, ESI, or Q-TOF mass
tively against 20 mM sodium phosphate buffer, pH 8.0, contain- spectrometry.
ing 100 mM NaCl and 50 mM L-arginine and then against the
Solid Phase Extraction—Purification of assay reaction prod-
same buffer lacking the NaCl, followed by the same buffer but ucts for analysis by MS was achieved by solid phase extraction
lacking the NaCl and arginine (this sequence of buffer changes on graphitized carbon (SPEGC) as described previously (19)
was found to be essential to retain PatB in solution; regardless, with some modifications. Reaction mixtures were added to
all buffer changes to a lower ionic strength led to the precipita- SPEGC columns that had been previously washed with one col-
tion of a fraction of the protein).
umn volume of acetonitrile (ACN) and then with three column
PatB was further purified by anion exchange chromatogra- volumes of water. Flow was achieved using a vacuum manifold
phy on Source Q. Samples were applied to the resin, previously (Phenomenex), and all eluted fractions were collected for anal-
equilibrated in 20 mM sodium phosphate buffer, pH 8.0, at a ysis. Elution of non-acidic sugars was achieved with 50% ACN,
flow rate of 1 ml⅐minϪ1, and the enzyme was recovered by whereas acidic sugars were recovered with 50% ACN in 0.1%
application of a linear gradient of 0–1.0 M NaCl over 50 min. trifluoroacetic acid (TFA). PG-derived muropeptides were
PatB eluted in ϳ100 mM NaCl. The purified protein was main- eluted with a 3:1 mixture of 2-propanol/ACN in 0.1% TFA,
tained at 4 °C and used immediately.
followed by 50% tetrahydrofuran to remove any remaining
Determination of Reaction Conditions for Optimal PatB bound material.
Activity—The dependence of the specific activity of PatBSUMO
Steady-state Kinetics—Routine kinetic assays of the O-
on pH was determined by assaying O-acetyltransferase activity acetyltransferase activity catalyzed by PatBSUMO involved incu-
16750 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of Peptidoglycan O-Acetyltransferase B
bation of the enzyme (1.0 or 3.0 ␮M) in 50 mM sodium phos- 300–2000 m/z in 4 GHz in extended dynamic range positive ion
phate buffer, pH 7, at 37 °C with 4 mM pNP-Ac (in 5% ethanol, mode. The instrument was externally calibrated with the ESI
final volume) and 1 mM chitopentaose as substrates. The pro- TuneMix (Agilent). For manual MS/MS of acetylated muro-
gress of reactions was monitored spectrophotometrically at 420 peptides, precursor ions were selected with an isolation width
nm for the release of p-nitrophenol (19). The rates of pNP-Ac of 1.5 m/z and a collision-induced dissociation reaction ampli-
hydrolysis (combined spontaneous and enzyme-catalyzed) tude of 1 V.
were obtained from negative control reactions that lacked
Other Analytical Techniques—Protein concentrations were
added chitopentaose, and they were subtracted from complete determined using a bicinchoninic acid assay (Pierce). SDS-
reaction rates (19). The ability of PatB to utilize other chromo- PAGE on 12% acrylamide gels was conducted by the method of
genic acetyl donors was investigated by substituting pNP-Ac Laemmli (25) with Coomassie Brilliant Blue staining and West-
with 0.2–5 mM ␣-naphthyl acetate (␣N-Ac; in 5% ethanol, final ern immunoblot analysis as described previously (19).
concentration) and 4-methylumbelliferyl acetate (MU-Ac; in
RESULTS
5% dimethyl sulfoxide, final concentration). The release of
␣-naphthol and 4-methylumbelliferone was monitored spec-
Production and Purification of PatB—Previously, we reported
trophotometrically at 320 and 355 nm, respectively. The release the production of a relatively soluble, truncated form of PatB
of co-enzyme A (CoA) from acetyl-, propionyl-, butyryl-, isobu- (PatB⌬36) lacking its N-terminal 36-amino acid signal sequence
tyryl, and hexanoyl-CoA was monitored using 5,5Ј-dithiobis(2- and membrane anchor, which we could purify in only modest
Ϫ1
nitrobenzoic acid) at 420 nm, and the production of O-acetyl yields (Ͻ1 mg⅐liter culture) (9). This enzyme was demon-
sugars was determined either by graphitized carbon HPLC or strated to function as PG O-acetyl-transferase in vivo, but
MS following reaction cleanup by SPEGC. The ability of PatB to efforts to use this enzyme in reproducible in vitro assays were
utilize different acceptors was investigated by substituting chi- problematic due to protein instability and precipitation despite
topentaose with other chitooligosaccharides (DP 2–6), a the inclusion of surfactants and other stabilizing agents in buf-
hexamer of chitosan (␤-(134)-linked glucosamine), cellooligo- fers. Given this, alternative protein constructs were sought.
saccharides (␤-(134)-linked glucose; DP 4 and 5), xylooligosac-
One of the major degradation products of the wild-type form
charides (␤-(134)-linked xylose; DP 4 and 6), and maltotriose of PatB produced from pACPM19 was a protein fragment with
(trimer of ␣-(134)-linked glucose).
an apparent molecular mass of 29.4 kDa as determined by
Michaelis-Menten parameters (kcat and Km) were deter- MALDI-TOF MS (data not shown). The missing segment was
mined by non-linear regression analysis of plots of initial veloc- determined to be from the N-terminal end of the protein based
ity as a function of substrate concentration. In all experiments, on Western immunoblot analysis showing the presence of the
n Ն 3.
C-terminal His6 tag (data not shown). It was hypothesized that
Mass Spectrometry—All MS analyses were conducted using this PatB fragment might be both active and stable because it
instruments at the Mass Spectrometry Facility of the Advanced would still possess the predicted catalytic residues or conserved
Analysis Centre, University of Guelph. MALDI-TOF MS was motifs (9). Thus, a new vector (pACPM30) was generated using
performed using 2,5-dihidroxybenzoic acid as a matrix for non- pACPM22 as a template, which provided a further truncation
amino sugars and 5-chloro-2-mercaptobenzothiazole for of the patB gene by 123 codons. The protein product, PatB
amino sugars or PG derivatives. Spectra were collected in pos- lacking its N-terminal 77 amino acid residues, PatB⌬77, was
itive mode on a Bruker Reflex III MALDI-TOF mass spectrom- purified by a combination of affinity and anion exchange chro-
eter in reflectron mode using a 337-nm nitrogen laser (set to matographies on Ni2ϩ-NTA-agarose and Source Q, respec-
Ϫ1
109–121 ␮J output). Statistical analyses were carried out using tively, with substantial yields (ϳ15 mg⅐liter
culture) and
the GraphPad Prism 5 software package.
relatively little apparent degradation (Fig. 1A). Preliminary
ESI-MS analyses were conducted with an Amazon SL ion activity assays performed with PatB⌬77 indicated that it pos-
trap mass spectrometer (Bruker Daltonics, Billerica, MA). Sam- sessed both esterase and transferase activity. However, con-
Ϫ1
ple was applied by direct infusion at a flow rate of 5 ␮l⅐min
.
cerns were raised over the impact of the 77-amino acid trunca-
The mass spectrometer electrospray capillary voltage was tion, and indeed, further analysis indicated that its transferase
maintained at 4.5 kV, and the drying temperature was set at activity was reduced as compared with PatB⌬26 (data not
300 °C with a flow rate of 8 liters⅐minϪ1. Nebulizer pressure was shown). Consequently, another alternative expression con-
40 p.s.i., and nitrogen was used as both nebulizing and drying struct was sought.
gas with helium used as collision gas at 60 p.s.i. The mass/
Recognizing its ability to stabilize the production of recom-
charge ratio was scanned across the m/z range 15–2,000 in binant proteins (27), PatB was engineered as a fusion protein
enhanced resolution positive ion mode. The resulting mass with SUMO. As with the generation of pACPM30 described
spectra were analyzed using the open source mMass software above, pACPM22 served as the template for this gene construc-
package (22–24).
tion. Consequently, the PatBSUMO product lacked the 26-
Q-TOF MS was performed on an Agilent UHD 6530 Q-TOF amino acid, periplasm-localizing signal sequence of PatB while
mass spectrometer. The flow rate was maintained at 0.2 possessing an N-terminal fusion to His6-SUMO. Again, the
ml⅐minϪ1, and the MS electrospray capillary voltage was main- combination of affinity and anion exchange chromatographies
tained at 4.0 kV with the drying gas temperature set to 350 °C was used to purify PatBSUMO to Ͼ95% homogeneity, as judged
with a flow rate of 13 liters⅐minϪ1. Nebulizer pressure was at by SDS-PAGE and MS analysis (Fig. 1B). However, efforts to
40 p.s.i. The mass/charge ratio was scanned across the range of remove the SUMO were met with difficulty with respect to
JUNE 13, 2014•VOLUME 289•NUMBER 24
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Characterization of Peptidoglycan O-Acetyltransferase B
the protein during its purification. Even with these precautions,
the half-life of the purified enzyme in 50 mM sodium phosphate
buffer, pH 7, at 25 °C was ϳ12 h, thus requiring that all assays be
conducted immediately after its preparation. Given this, an
accurate measurement of specific activity for the liberated PatB
was impossible to obtain.
Determination of Assay Conditions—The dependence of
PatBSUMO activity on pH was assessed by determining its spe-
cific activity as an O-acetyltransferase in different buffers rang-
ing from pH 4 to 9 using chitotriose and pNP-Ac as co-sub-
strates with the porous graphitized carbon HPLC-based assay.
Under the conditions employed, the activity profile was rela-
tively broad and bell-shaped with maximal activity at pH 7.0
(Fig. 1C). No activity was detected at pH 4.0. The bell shape of
the plot suggests that at least two ionizable groups on the
enzyme are essential for catalytic activity (both the acetyl donor
and acceptor substrates used in this assay are uncharged).
The addition of the divalent metals tested (viz. Mg2ϩ, Ca2ϩ
,
Mn2ϩ, Fe2ϩ, Cu2ϩ, and Zn2ϩ) was found to be detrimental to
PatBSUMO activity, with most leading to the precipitation of the
protein. Although the addition of CaCl2 and MgCl2 did not
appear to precipitate the protein, they were found to decrease
specific activity by 43 and 80%, respectively, compared with
control enzyme. The inclusion of 5 mM EDTA during purifica-
tion steps had little impact on specific activity (increase by
Ͻ5%), indicating that metals are unlikely to be involved in catal-
ysis. The addition of 100 ␮g⅐mlϪ1 BSA reduced specific activity
by 34%, whereas 50 mM NaCl resulted in a decrease of 21%.
Given these data, all further routine assays for activity were
conducted using a simple buffer of 50 mM sodium phosphate,
pH 7.0.
Specificity of PatBSUMO for Different Acetyl Donors—Despite
the proposed role of the integral membrane protein PatA as the
translocator and donor of acetyl groups to PatB for the O-acety-
lation of PG in vivo (9, 10), PatB was recently discovered to
utilize the simple activated acetyl substrates acetyl-CoA and
pNP-Ac for in vitro transacetylations (19). The assay that was
subsequently developed exploits the chromogenic properties of
the liberated p-nitrophenylate co-product as the acetyl group is
transferred from pNP-Ac to an acceptor co-substrate.
FIGURE 1. Purification and dependence of PatB activity on pH. A, Coomas-
sieBrilliantBluestainedSDS-polyacrylamidegeloffractionstakenatdifferent
points during the purification of PatB⌬77 by immobilized metal affinity chro-
matography (IMAC) and anion exchange chromatography on Source Q. The E
and SQ fractions were purposely overloaded to demonstrate the respective
presence and absence of low abundance contaminants. WC, whole cell frac-
tion; FT, flow-through fraction; W1 and W2, wash 1 and 2, respectively; E,
fractionelutedwithimidazole;SQ, fractionfromSourceQchromatography. B,
Coomassie Brilliant Blue-stained SDS-polyacrylamide gel of fractions taken at
different points during the purification of PatBSUMO. The expected molecular
mass of PatBSUMO is 46,532.5 Da. UC, uninduced whole cell fraction; IC,
inducedwholecellfraction. Themigrationofmolecularmassmarkers(inkDa)
is presented on the left of each panel. C, PatBSUMO activity was assayed by
graphitized carbon HPLC using acetyl-CoA and chitotriose as co-substrates in
50 mM ammonium acetate (E), 50 mM sodium phosphate (f), and 50 mM
Tris-HCl (ࡗ) at the pH values indicated. Error bars, S.D. (n Ն 2).
The possibility that other chromogenic substrates may serve
as better co-substrates for PatB was investigated by monitoring
hydrolytic reactions. Thus, as with pNP-Ac, the differences
between the rates of spontaneous and PatBSUMO-catalyzed
hydrolysis of ␣N-Ac and MU-Ac were determined with varying
maintaining PatB stability. First, the removal of SUMO using concentrations of the chromogens. As seen in Table 2, the
SUMO protease resulted in the production of several PatB frag- enzyme was most efficient with pNP-Ac as substrate, as
ments that could not be resolved from one another. This was reflected by the determined kcat/Km values. Consequently, this
surprising, given the reported specificity of the SUMO protease chromogen was used for all further kinetic analyses.
(27), so the limited digestion observed probably occurred spon-
To evaluate the ability of PatBSUMO to utilize other acyl
taneously following cleavage of SUMO from PatB (data not groups as substrate, the hydrolysis of propionyl-, butyryl-,
shown). Second, the removal of SUMO led to significant pre- isobutyryl, and hexanoyl-CoA was tested. Of these, the enzyme
cipitation of protein, resulting in very low protein yields. As was only able to utilize propionyl-CoA. A kinetic analysis of this
before, repeated efforts to stabilize PatB by inclusion of surfac- hydrolysis provided a Km value of 2.9 mM, which is only 3-fold
tants, different buffer salts, and glycerol were unsuccessful. higher than that for Ac-CoA, and a kcat value 2.6-fold lower
Given these issues, SUMO had to be retained on the recombi- (Table 2). Overall, however, these data indicate almost an order
nant PatB for its biochemical and kinetic characterization, and of magnitude difference in catalytic efficiency between the two
significant efforts were still required to prevent precipitation of CoA substrates. Nonetheless, PatBSUMO was able to transfer
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Characterization of Peptidoglycan O-Acetyltransferase B
the proprionyl to an acceptor sugar, such as chitooligosaccha- tive of cellohexaose, chitosan (DP ϭ 6), was tested as a potential
rides (Fig. 2).
acceptor substrate. On the other hand, and as previously
Acceptor Substrate Specificity—Various commercially avail- described (19), PatBSUMO did catalyze the transfer of acetyl to the
able oligosaccharides were tested under standard reaction con- N-acetyled derivatives of chitosan oligomers, chitooligosaccha-
ditions to identify the minimal acceptor substrate requirements rides with DP Ն 3; no product was found for the disaccharide
for PatBSUMO in vitro. As expected, the enzyme was unable to chitobioseasanalyzedforbyMS(Table3). Atextendedincubation
transfer the acetyl to oligomers (DP ϭ 4 and 6) of xylan, times (Ͼ10 min), reaction products were observed with multiple
homopolymers of D-xylose, which thus lack a C-6 hydroxyl; the acetylations, although the number of modifications to an individ-
natural O-acetylation of the C-3 hydroxyl of xylan occurs in ual oligosaccharide was always less than its DP (19).
plants (26). However, polymers of glucose (DP ϭ 4 and 6),
which differ from xylose by the presence of a C-6 and its corre-
TABLE 3
sponding hydroxyl moiety, also did not serve as a substrate. Oligosaccharide substrate specificity of PatBSUMO
Potential substrates in 50 mM sodium phosphate buffer, pH 6.5, containing 4 mM
Likewise, no acetylated product was observed in MALDI-TOF
pNP-Ac were incubated with 1.0 ␮M PatBSUMO, and samples of reaction mixtures
MS spectra of reaction mixtures when the amino sugar deriva-
were subjected to MALDI-TOF MS analysis. ND, not detected.
m/z͓m؉Na͔of potential
O-acetylated product
Theoretical Detected Difference
TABLE 2
Potential substrate
Kinetic parameters of esterase and O-acetyltransferase activities of
PatBSUMO
m/z
m/z
PG metabolites
Substrate
Km
mM
kcat
s
kcat/K
m
Ϫ1 Ϫ1
GlcNAc
286.09
316.10
561.19
759.28
ND
ND
ND
ND
Ϫ1
MurNAc
M
⅐s
GlcNAc-MurNAc
GlcNAc-MurNAc-dipeptide
Hydrolysis activitya
Ac-CoA
0.99 Ϯ 0.08
2.9 Ϯ 0.50
5.5 Ϯ 2.7
0.011 Ϯ 0.0003
0.0043 Ϯ 0.00033
0.34 Ϯ 0.15
11 Ϯ 0.90
1.5 Ϯ 0.31
62 Ϯ 40
Propionyl-CoA
␣N-Ac
Chitooligosaccharides
GlcNAc2
489.17
579.22
692.25
895.33
1,098.41
1,301.49
ND
MU-Ac
0.44 Ϯ 0.04
0.96 Ϯ 0.10
0.11 Ϯ 0.004
0.76 Ϯ 0.032
260 Ϯ 26
797 Ϯ 88
GlcNAc2-O-benzyl
GlcNAc3
579.45
692.45
894.54
1098.85
1302.18
0.23
0.20
pNP-Acb
GlcNAc4
Ϫ0.79
0.44
Transferase activityc
GlcNAc2
GlcNAc5
GlcNAc6
0.69
GlcNAc2-O-benzyl
b
GlcNAc3
2.63 Ϯ 0.22
1.84 Ϯ 0.14
0.95 Ϯ 0.10
0.73 Ϯ 0.05
0.74 Ϯ 0.02
0.52 Ϯ 0.01
0.65 Ϯ 0.02
0.58 Ϯ 0.02
280 Ϯ 25
283 Ϯ 23
681 Ϯ 74
791 Ϯ 59
Other oligosaccharides
␤-1,4 Glc6
␤-1,4 Glc4
␣-1,4 Glc3
Xylose6
GlcNAc4
GlcNAc5
GlcNAc6
1,055.33
731.22
569.17
875.26
611.18
1,049.42
ND
ND
ND
ND
ND
ND
a Reactions conducted in 50 mM sodium phosphate buffer, pH 6.5, at 37 °C.
b Data from Ref. 19.
Xylose4
c Same reaction conditions with 4 mM pNP-Ac serving as acetyl donor and 0.2–8
mM chitooligosaccharides as acceptors (0.2–2.5 mM chitohexaose).
GlcN6
FIGURE 2. MALDI-TOF MS analysis of PatB reaction products with propionyl-CoA as donor substrate. Enzyme (1.0 ␮M) in 50 mM sodium phosphate buffer
(pH 6.5) was incubated with 4 mM propionyl-CoA and chitotetraose at 25 °C. After 30 min of incubation, samples were desalted using SPEGC and applied to a
Bruker Reflex III mass spectrometer using 5-chloro-2- mercaptobenzothiazole as the matrix. Contamination of chitotetraose with chitooligosaccharides of
other DPs (as supplied by the manufacturer) is apparent; all identified species are sodium adducts. a.i., absolute intensity.
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Characterization of Peptidoglycan O-Acetyltransferase B
FIGURE 3. Identification of initial PatBSUMO reaction products. PatBSUMO (1 ␮M) in 50 mM sodium phosphate buffer, pH 7.0, was incubated with 4 mM Ac-CoA
and 1 mM chitotetraose (A) and chitohexaose (B). Following 1 min at 25 °C, reactions were quenched by acidification, and products were subjected to
adsorption HPLC on graphitized carbon. The column was equilibrated in water, and a linear gradient to 100% acetonitrile (1%/min for chitotetraose; 2%/min
for chitohexaose) was applied to elute solutes. Fractions of interest were collected, dried under vacuum, and resuspended in 100 ␮l of 50:50 water/methanol
for Q-TOF MS analysis in positive ion mode. C, Q-TOF-MS analysis of PatBSUMO O-acetyltransferase activity with 1 mM chitopentaose and 4 mM pNP-Ac at 25 °C.
After 1 min of incubation, 10 ␮l of the reaction mixture was directly infused into the mass spectrometer. The expected positions of the (absent) doubly and
triply acetylated species are also denoted. mAU, milliabsorbance units; a.i., absolute intensity.
FIGURE 4. O-Acetyltransferase activity of PatBSUMO on solubilized PG-derived muropeptides as detected by ESI-ion trap MS. pNP-Ac (1 mM) in 50 mM
sodium phosphate buffer, pH 7.0, and 2 mM PG, previously solubilized by treatment with hen egg white lysozyme and E. coli PBPs 4 and 7 in the absence (A) and
presence (B) of H. pylori Csd6, were incubated in the absence (blue) and presence (red) of PatBSUMO (1 ␮M). After mixing for 10 min at 25 °C, samples were
withdrawn, desalted by SPEGC, and subjected to ESI-ion trap MS analysis. r. int., relative intensity.
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Characterization of Peptidoglycan O-Acetyltransferase B
TABLE 4
To determine the nature of the initial reaction products of
Muropeptide products of PatBSUMO activity on PG identified by ESI -
ion trap MS
PatBSUMO acting on chitooligosaccharides, samples of reaction
mixtures incubated for 1 min were infused directly into the
Q-TOF mass spectrometer for analysis. As depicted in Fig. 3 for
the reaction with chitopentaose, a single product peak was
detected with an m/z consistent with singly acetylated chitope-
ntaose; no products were observed at the expected m/z for mul-
tiple acetylated species. Nonetheless, separation of reaction
products by adsorption chromatography on graphitized carbon
indicated that PatBSUMO produced two structurally distinct ini-
tial products (Fig. 3). To probe this apparent discrepancy, frac-
tionated products from reactions with chitotriose, chitote-
traose, and chitohexaose as substrates were collected and
subjected to ion trap MS/MS. These analyses indicated that
indeed two unique but singly acetylated products were pro-
duced and resolved by the graphitized carbon HPLC column.
With each substrate, the difference between the two reaction
products was the positioning of the transferred acetyl. Thus,
with chitotetraose and chitohexaose, acetylations occurred
exclusively at either the third or fourth residues of the respec-
tive oligomers, whereas only the first or second residue of chi-
totriose served as acceptor sites.
PG digested with lysozyme and E. coli PBPs 4 and 7 in 50 mM sodium phosphate
buffer, pH 7.0, was incubated with 4 mM pNP-Ac in the absence and presence of
1 ␮M PatBSUMO and subjected to MS analysis. Muropeptides listed in black were
present in both reaction product mixtures, whereas those in red were unique to
reactions in the presence of PatBSUMO
.
Assay of PG O-Acetylation in Vitro—A variety of commercially
available PG-related metabolites were tested as acceptors for Pat-
BSUMO in vitro. These included the monosaccharide MurNAc, the
disaccharide GlcNAc-␤-1,4-MurNAc, and its dipeptide derivative
GlcNAc-␤-1,4-MurNAc-l-Ala-D-iso-Gln. No O-acetyl product
was observed for any of these sugars, further demonstrating the
enzyme’s need for oligomers of carbohydrate with DP Ն 3 (as seen
with the use of chitooligosaccharides). Unfortunately, such PG-
related metabolites are not commercially available.
With the lack of commercial products, we prepared a pool of
soluble muropeptides (viz. GlcNAc-MurNAc peptides) by
digesting insoluble PG with a mixture of mutanolysin (a mur-
amidase) and PBPs 4 and 7 (peptidases) from E. coli. Initial
O-acetyltransferase reactions with this heterogeneous mixture
of solubilized muropeptides resulted in precipitation of
PatBSUMO, probably due to buffer components (MgCl2, NaCl,
etc.) that were required to stabilize the hydrolases used for the
prior PG digestion. Consequently, we developed a novel isola-
tion procedure to rapidly desalt and concentrate the solubilized
muropeptides. Taking advantage of the high binding capacity of
SPEGC, adsorption of the muropeptides to the graphitized car-
bon and elution in volatile solvent successfully removed buffer
salts and resulted in the production of a pool of diverse and
highly concentrated muropeptides as characterized by ESI-MS
and quantified by high performance anion exchange chroma-
tography (data not shown). This protocol provided concentra-
tions of muropeptides as high as 26 mM (as determined by
MurNAc content) that involved mixtures of di-, tri-, and tetra-
peptide stems (GM2, GM3, and GM4, respectively, where G
and M represent GlcNAc and MurNAc, respectively) but pre-
dominantly muro-tetrapeptides (GM4).
TABLE 5
Muropeptide products of PatBSUMO activity on PG enriched with
muro-tripeptides identified by ESI - ion trap MS
PG digested with lysozyme, E. coli PBPs 4 and 7, and H. pylori Csd6 in 50 mM sodium
phosphate buffer, pH 7.0, was incubated with 4 mM pNP-Ac in the absence and
presence of 1 ␮M PatBSUMO and subjected to MS analysis. Muropeptides listed in
black were present in both reaction product mixtures, whereas those in red were
unique to reactions in the presence of PatBSUMO
.
Using final concentrations of 2 mM, PatBSUMO was observed
to transfer acetyl groups to these muropeptides (Fig. 4A and prising given the enrichment of the corresponding substrates in
Table 4). Further MS analysis of these products indicated the the PG digests. Importantly, similar results were obtained with
predominance of acetylated muro-tetrapeptides, both di- and PatB liberated from SUMO by prior digestion with SUMO pro-
tetrasaccharides (GM4 and GM4-GM4), which was not sur- tease, although the instability of this form of the enzyme
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Characterization of Peptidoglycan O-Acetyltransferase B
required significantly higher concentrations to be used (viz. 5
␮M PatB versus 1 ␮M PatBSUMO). To investigate further this
apparent specificity for muro-tetrapeptides, the soluble muro-
peptide pool was digested with Helicobacter pylori Csd6, a car-
boxypeptidase with specificity for the terminal D-Ala of these
metabolites (28). MS analysis of this digestion confirmed that
the resulting pool of muropeptides was almost exclusively com-
posed of muro-tripeptides (both di- and tetrasaccharides; GM3
and GM3-GM3). Unlike with GM4, PatBSUMO did not transfer
the acetyl to GM3 (Fig. 4B and Table 5). However, the dimer
GM3-GM3 did serve as an acceptor for the enzyme because a
single O-acetylation was observed. It should be noted, however,
that even in the presence of high concentrations of substrate,
this activity was relatively weak.
Ion trap MS/MS was used again to confirm both the identity
the reaction products of reactions with the tetrasaccharide sub-
strates and the positioning of the transferred acetyl specifically
to muramoyl residues (Fig. 5), as occurs naturally on the PG of
N. gonorrhoeae. These analyses also revealed that the acetyla-
tions occurred on the internal MurNAc residues of the tetra-
saccharides, thus confirming specificity for the C-6 hydroxyl
group; this internal MurNAc does not have another available
hydroxyl moiety for acetylation. Finally, there was no evidence
of O-acetylated GlcNAc in the reaction products.
FIGURE 6. Example assays of O-acetyltransferase activity of PatB. A,
PatBSUMO (1 ␮M) in 50 mM sodium phosphate buffer, pH 7.0, was incubated at
25 °C with 3 mM pNP-Ac hydrolysis in the presence (F) and absence (E) of 2
mM chitopentaose. The rates of p-NP production were monitored spectro-
photometrically at 410 nm, and O-acetyltransferase activity was determined
Steady State Kinetics of PatBSUMO—Our recently developed
chromogenic assay (19) was employed to determine the
Michaelis-Menten parameters for the O-acetylation of chito-
oligosaccharides by PatBSUMO. This assay monitors the release by the difference between the two. B, initial rates of reaction of PatBSUMO
incubated with 4 mM pNP-Ac and varying concentrations of chitopentaose.
Error bars, S.D. (n ϭ 3).
of p-nitrophenol from pNP-Ac as the acetyl group is transferred
to either water or an acceptor chitooligosaccharide; transacety-
lase activity is calculated from the difference between the two
not been possible due to the significant technical limitations asso-
ciated with the complexity of attempting to analyze a peripheral
membrane protein that receives its acetyl co-substrate from a
transmembrane protein, which it then donates to a completely
insoluble heteropolymer. As presented herein, we were able to
overcome these limitations by using a chemical biological
approach. In an earlier study, we demonstrated that PatB is capa-
ble of catalyzing transfer of acetyl from pNP-Ac (19), thus dispens-
ingwiththeneedforthenaturalpartnerofPatB, theintegralmem-
brane protein and putative translocator of acetyl, PatA (13, 19). In
the current study, we were able to expand upon this and kinetically
characterize the family of acetyl donors and acceptors that PatB is
able to utilize. The pool of donors includes the non-O-linked
rates. Representative plots of the hydrolysis of pNP-Ac in the
absence and presence of acceptor ligand are presented in Fig.
6A. Initial rates of activity were determined at pH 7, holding the
concentration of pNP-Ac constant at 4 mM, a concentration
that is ϳ4 times Km (and just below the solubility limit of the
chromogen) (Fig. 6B). Values of kcat were similar for the series
of chitooligosaccharides tested, whereas Km values decreased
slightly from chitotriose through to chitohexaose, with the
greatest difference occurring between chitotetraose and chito-
pentaose (Table 2). This difference is more apparent in the cal-
culated efficiency constants kcat/Km.
DISCUSSION
We report herein the first kinetic analysis of PatB, a major acetyl-CoA in addition to two other O-linked donors (MU-Ac and
virulence determinant found in numerous bacterial pathogens, ␣N-AC), suggesting significant active site plasticity. MU-Ac is a
both Gram-positive and Gram-negative (5, 17, 29, 30), and a fluorogenic substrate/acetyl donor, and as such, it will serve as an
potential new target for antibacterial therapy. Our elucidation exceptional tool for high throughput screening of this family of
of the substrate profile for PatBSUMO in vitro provides valuable enzymes, given that many inhibitory compounds are often not
insight for the development of an assay amenable to the high amenable to colorimetric assays.
throughput screening for inhibitors that may serve as new leads
for antibiotic development.
The finding that PatB is able to catalyze transfer from thio-
esters was surprising, given the absence of acetyl-CoA in the
Despite being identified almost 10 years ago (10, 11, 13), a periplasm/extracellular space of bacteria. This apparent pro-
kinetic analysis of the enzymes involved in PG O-acetylation had miscuity of acetyl-donor specificity is consistent with our ear-
FIGURE 5. Ion trap MS/MS analysis of O-Ac-GM4-GM4 (A) and O-Ac-GM3-GM3 (B) identified in Fig. 4. The MS/MS spectrum of m/z ϭ 952.43 (of Fig. 4A) and
m/z ϭ 881.18 (of Fig. 4B) indicates that the PatBSUMO reaction products are O-acetylated on the C-6 hydroxyl moiety of MurNAc on the non-reducing MurNAc
residue for both tripeptide- and tetrapeptide-containing species.
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Characterization of Peptidoglycan O-Acetyltransferase B
FIGURE 7. Proposed pathway for the metabolism of PG in the periplasm. Acetyl (Œ) is translocated from the cytoplasm to the periplasm by PatA for
PatB-catalyzed transfer to MurNAc residues in PG possessing tetrapeptide stems (previously generated by PBP-catalyzed transpeptidation) (step 1). The
carboxypeptidase (CP) activity of a PBP trims the tetrapeptide stem of O-acetyl-MurNAc residues to a tripeptide (step 2), providing the appropriate substrate for
O-acetyl-PG esterase, Ape1. Ape1 catalyzes the de-O-acetylation of PG (step 3), thereby exposing MurNAc residues to cleavage by lytic transglycosylases, which
generate/release 1,6-anhydro-MurNAc products (step 4).
lier observation that PatB was able to receive acetyl groups from production of over 70 distinct muropeptides, as first demon-
WecH (9), an integral membrane protein involved in the strated by Glauner (32). In our search for a more consistent and
O-acetylation of enterobacterial common antigen in E. coli. soluble co-substrate for in vitro studies, we found that the min-
Such flexibility for the relatively small acetyl donors may not be imum requirements were for oligomers of GlcNAc with a DP Ն
too surprising, however, when considering that the proposed 3. Chitooligosaccharides are simple structural mimics of PG
natural source of acetyl for PatB is acetyl-bound PatA. If indeed oligomers, and that they serve as pseudosubstrates for PatB is
this postulate is correct, and that there is no intermediary car- analogous to the ability of lysozyme to also utilize them as sub-
rier molecule involved in the transfer process between the two strate, albeit weakly (33, 34). The observation that the DP of the
proteins, PatB must complex with PatA and thus have a binding chitooligosaccharides had to be Ն3 to serve as acceptor for PatB
site/cleft to accommodate both the binding elements of PatA is consistent with early reports concerning the O-acetylation of
and its linked acetyl. Regarding a PatA-PatB complex, its for- PG in N. gonorrhoeae, Proteus mirabilis, and S. aureus. These
mation, involving the reaction centers of both proteins, would earlier biochemical studies demonstrated that O-acetylation
probably preclude free water from accessing the active site and occurs shortly after transglycosylation and transpeptidation of
thereby minimize, if not prevent, the hydrolysis of the translo- Lipid II (undecaprenyl-bound GlcNAc-MurNAc-pentapeptide)
cated acetyl group that we observe in vitro (and exploited (29, 35–39), thus involving PG chains and not the precursor disac-
herein). This exclusion of water would thus ensure the effi- charide. The modest improvement in the Km and specificity con-
ciency of the O-acetylation process that occurs outside of the stant for the chitooligosaccharides with increasing DP to 5/6 sug-
cytoplasm and prevent the wasteful loss of acetate.
gests that PatB, like many other carbohydrate-active enzymes,
That PatB may have a relatively flexible active site was further including lysozyme (40), probably has several sugar binding sub-
exemplified by the finding that it could transfer propionyl sites in its active site. The multiacetylations observed with the chi-
groups to acceptors in addition to acetyls, albeit at a much tooligosaccharides would reflect the relative structural simplicity
reduced rate. Recently, the modification of muropeptides for of the substrate because presumably it is able to bind at these sub-
use as adjuvants has been explored (reviewed in Ref. 31). One of sites in different orientations.
the limiting factors in this research is the relatively low lipophi-
Despite the complexities of working with PG-derived
licity of these molecules. The capacity of PatB to transfer pro- ligands, a clear specificity of PatB was observed in vitro with
pionyl groups to oligosaccharides could be further exploited these more complex acceptor substrates, where a preference
through reaction optimization and perhaps protein engineer- was found for any muropeptides possessing tetrapeptides or
ing to make possible the transfer of longer-chain acyl groups.
longer chain muropeptides (DP Ն 2) with tripeptide stems.
Initial studies with PG digestion products as acceptor sub- This again is consistent with the earlier in vivo studies indicat-
strate led to our collection of inconsistent data, probably due to ing that O-acetylation follows transpeptidation of growing PG
the heterogeneous nature of the polymer. Indeed, muramidase chains (29, 35–39); the formation of cross-links in PG by trans-
digests of insoluble PG from a single bacterial source lead to the peptidases results in the release of the terminal D-Ala from stem
16758 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of Peptidoglycan O-Acetyltransferase B
REFERENCES
pentapeptides, leaving tetrapeptides on muramyl residues.
Whereas high resolution data are not available for the structure
of all muropeptides generated by N. gonorrhoeae, the apparent
specificity of PatB for muro-tetrapeptides is also consistent
with the known PG composition of closely related N. meningi-
tidis, which encodes homologs of PatA and PatB, both with 96%
identity to the N. gonorrhoeae proteins and with complete con-
servation of the consensus motifs (19). Analysis of N. meningi-
tidis PG has indicated that O-acetylation is found predomi-
nantly on muro-tetrapeptides (41, 42), and a phenotypic study
involving mutants deficient in O-acetylation suggested that the
modification occurred mostly on muropeptides containing
tetra- or pentapeptide stems (30). Given the relatedness of
N. gonorrhoeae, it is likely that its PG possesses a similar com-
position, but this, together with a more detailed analysis of PatB
substrate specificity in vivo, will form the basis of a future study.
A model for the role of O-acetylation in regulation of PG chain
lengthinN. meningitidishasrecentlybeenproposed. Accordingto
Veyrier et al. (30), the O-acetyl-PG esterase Ape1 only deacetylates
MurNAc residues containing tripeptide stems in vivo. They pro-
pose that newly deacetylated muro-tripeptides serve as substrate
for a lytic transglycosylase, thus limiting PG chain length. Consis-
tent with this model, our data show that PatB has specificity for
muro-tetrapeptides in vitro, thereby preserving these regions of
PG from lytic activity. If the model of Veyrier et al. (30) is correct,
it would be expected that a carboxypeptidase (PBP?) with specific-
ity for O-acetyl-MurNAc-tetrapeptide would cleave the tetrapep-
tide as required, thereby generating the tripeptide substrate for
Ape1, which in turn releases the O-acetyl group to provide an
accessible lytic transglycosylase substrate (Fig. 7). A zymographic
method for determining substrate specificity of lytic enzymes has
been developed (43), but unfortunately it has not yet been applied
for the detection of O-acetyl-PG-specific enzymes from
N. gonorrhoeae.
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Chapot-Chartier, M.-P. (2011) Characterization of O-acetylation of N-
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In this study, we have demonstrated that, in vitro, PatB is pref-
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eties or muroglycans containing tetrapeptide stems. This informa-
tion will be valuable for the identification and development of PG
O-acetyltransferase inhibitors that could represent potential leads
to novel classes of antibiotics that are desperately needed by clini-
cians combating bacterial infections and diseases. Indeed, in its
2013 Threat Report, the Centers for Disease Control and Preven-
tion listed drug-resistant N. gonorrhoeae, together with carbap-
enem-resistant Enterobacteriaceae and Clostridium difficile, as its
three urgent (highest level) threats in the United States (44).
Finally, this study will make possible, for the first time, in vitro
characterization of other related acetyltransferases, yielding
important insights into the role of glycan modification in extracel-
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Acknowledgments—We thank K. Young (University of Arkansas
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