Characterization of wall teichoic acid degradation by the bacteriophage φ29 appendage protein GP12 using synthetic substrate analogs

Article abstract of DOI:10.1074/jbc.M115.662866

Background: The GP12 protein from bacteriophage φ29 is a likely wall teichoic acid hydrolase. Results: GP12 is an efficient catalyst of wall teichoic acid hydrolysis that is influenced by glycosylation of the wall teichioc acid polymer. Conclusion: GP12 m

Full text of DOI:10.1074/jbc.M115.662866

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 31, pp. 19133–19145, July 31, 2015
2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
©
Characterization of Wall Teichoic Acid Degradation by the
Bacteriophage ␸29 Appendage Protein GP12 Using Synthetic
□
S
*
Substrate Analogs
Received for publication, May 5, 2015, and in revised form, June 15, 2015 Published, JBC Papers in Press, June 17, 2015, DOI 10.1074/jbc.M115.662866
‡1
‡
‡
§
Cullen L. Myers , Ronald G. Ireland , Teresa A. Garrett , and Eric D. Brown
‡
From the Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research,
McMaster University, Hamilton, Ontario L8N 3Z5, Canada and the Department of Chemistry, Vassar College,
§
Poughkeepsie, New York 12604
Background: The GP12 protein from bacteriophage ␸29 is a likely wall teichoic acid hydrolase.
Results: GP12 is an efficient catalyst of wall teichoic acid hydrolysis that is influenced by glycosylation of the wall teichioc acid
polymer.
Conclusion: GP12 may serve as a tool for the characterization of wall teichoic acid polymers.
Significance: Characterization of a prototypical wall teichoic acid hydrolase using chemically defined substrates.
The genetics and enzymology of the biosynthesis of wall biotics (2–4), revealing a surprising role for WTA in ␤-lactam
teichoic acid have been the extensively studied, however, com- resistance and highlighting steps in its biosynthetic pathway as
paratively little is known regarding the enzymatic degradation attractive antibacterial therapeutic targets.
of this biological polymer. The GP12 protein from the Bacillus
subtilis bacteriophage ␸29 has been implicated as a wall teichoic
acid hydrolase. We have studied the wall teichoic acid hydrolase
activity of pure, recombinant GP12 using chemically defined
wall teichoic acid analogs. The GP12 protein had potent wall
teichoic acid hydrolytic activity in vitro and demonstrated ϳ13-
fold kinetic preference for glycosylated poly(glycerol phos-
phate) teichoic acid compared with non-glycosylated. Product
distribution patterns suggested that the degradation of glycosy-
lated polymers proceeded from the hydroxyl terminus of the
polymer, whereas hydrolysis occurred at random sites in the
non-glycosylated polymer. In addition, we present evidence
that the GP12 protein possesses both phosphodiesterase and
phosphomonoesterase activities.
The genetics and biochemistry of WTA biosynthesis have
been extensively studied in both the model Gram-positive bac-
terium Bacillus subtilis and the pathogen S. aureus (1). Biosyn-
thesis of poly(glycerol phosphate) WTA in B. subtilis is illustra-
tive (see Scheme 1). Polymer synthesis begins on the inner cell
surface with the addition of N-acetlyglucosamine-1-phosphate
to membrane-bound undecaprenyl phosphate, catalyzed by the
TagO enzyme and generating lipid ␣ (WTA intermediates are
named after the enzymes that act on them (5), summarized in
Table 1). Next, TagA adds N-acetlymannosamine to lipid ␣ to
form lipid ␤, the substrate for the primase enzyme TagB that
adds a single glycerol phosphate molecule to form lipid ␸.1 (6).
TagF is the polymerase that adds 30 to 50 glycerol phosphate
units to this substrate (7), followed by the modification of mul-
tiple glycerol units in poly(glycerol phosphate) WTA with
␣
-glucose by TagE (8). The polymer is then transported to the
2
Wall teichoic acids (WTAs) are polyanionic glycopolymers
extracellular surface by the TagGH ABC transporter system (9)
where the gene products of the dltABCD operon effect D-ala-
lynation (10) and the tagTUV enzymes are believed to catalyze
final polymer transfer to N-acetylmuramic acid of peptidogly-
can (11).
covalently attached to peptidoglycan in the cell wall of Gram-
positive bacteria. Prototypical WTA polymers are repeating
units of 1–3-linked glycerol phosphate or 1–5-linked ribitol
phosphate and constitute roughly 50% of the wall mass (1).
WTAs have been associated with diverse cellular processes
including cell division and morphology, peptidoglycan assem-
bly, bacteriophage recognition and adherence, and as a viru-
lence determinant (1). Recently, the disruption of WTA
biosynthesis was shown to restore the susceptibility of methi-
cillin-resistant Staphylococcus aureus to certain ␤-lactam anti-
In contrast to the thorough understanding of its biosynthesis,
the enzymology and biological relevance of WTA degradation
have not been well studied. Evidence of such enzymatic activity
was first reported more than 40 years ago by Wise and co-work-
ers (12), with the discovery that B. subtilis is capable of utilizing
its cell wall as a phosphate source. This was attributed to
expression of a so-called teichoicase with glycerophosphodies-
*
This work was supported by a Canada Research Chair award (to E. D. B.) and terase activity specific for WTA. It was later speculated that the
Canadian Institutes for Health Research Grant MOP-15496. The authors
declare that they have no conflicts of interest with the contents of this
article.
S
expression of this enzyme may be induced by phosphate star-
vation (13), enabling the procurement of necessary phosphate
from WTA. In line with this, during phosphate-limited growth
of B. subtilis, much of the WTA in the cell wall is replaced by the
phosphate-free polymer teichuronic acid (14, 15). Apart from
the potential importance in the phosphate starvation response,
□
This article contains supplemental Figs. S1 and S2 and Table S2.
To whom correspondence should be addressed. Tel.: 905-525-9140 (ext.
1
2
2454); Fax: 905-222-9033; E-mail: ebrown@mcmaster.ca.
2
The abbreviations used are: WTA, wall teichoic acid; bis-pNPP, bis-para-ni-
trophenyl phosphate; pNP, para-nitrophenol.
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Wall Teichoic Acid Degradation by GP12
Lipid φ.1 analog
SCHEME 1. Synthesis of tridecyl-linked WTA analogs.
TABLE 1
Nomenclature and chemical composition of analogs for wall teichoic acid precursors and polymers
Analog
Chemical compositiona
Lipid ␣ analog
GlcNAc-1-P-P-tridecane
Lipid ␤ analog
ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane
GroP-ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane
Lipid ␸.1 analog
Lipid ␸.n analog
(GroP) -ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane
n
Glycosylated lipid ␸.n analog
(␣-D-Glu-(132)-GroP) -ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane
n
a
P, phosphate; GlcNAc, N-acetyl glucosamine, ManNAc, N-acetyl mannosamine; GroP, glycerol 3-phosphate; Glu, glucose.
a role for WTA degradation in the regulation of cell wall struc- gesting that the protein has inherent wall teichoicase activity.
ture and composition can be envisioned. For instance, WTAs The activity was noted to be highly dependent on divalent metal
2
ϩ
2ϩ
have been shown to direct bacterial peptidoglycan hydrolases ions, two of which (Ca and Mg ) were observed bound at
(
16–19) and consequently, influence wall remodeling. Also, as the proposed WTA-degrading active site in a pectin lyase
much as 50% of the wall is recycled during bacterial cell division domain (28), prompting the suggestion of a two-metal ion
20, 21), implying a need perhaps for enzymes capable of pro- mechanism for catalysis of WTA phosphodiester bond hydro-
(
cessing the very large quantities of WTA in peptidoglycan.
lysis (27). Analogous mechanisms for phosphate ester hydroly-
Through activity-guided purification, Kusser and Fielder (22, sis involving a bimetallated active center are known for
3) achieved some enrichment of the wall teichoicase activity enzymes from the binuclear metallophosphatase family, which
2
from B. subtilis and later from Bacillus pumilus (24), yet these includes glycerophosphodiesterases and purple acid phospha-
studies were limited in two important aspects. First, the enzyme tases (29), and interestingly, GP12 is structurally unrelated to
preparations were never linked to a gene or protein sequence. this family.
Second, the substrates used were poorly defined, consisting of
In light of the previous qualitative examination, GP12 repre-
crude cell wall preparations or partially purified WTAs. Thus, sents an ideal candidate for the study of enzyme-catalyzed
bacterial teichoicases remain obscure, as are the mechanistic WTA degradation. To that end, we have performed in-depth
details of their biochemical activity.
investigations on this prototypic wall teichoicase (GP12) using
Although bacterial enzymes that degrade WTA and their chemically defined and soluble WTA substrate analogs. Indeed,
corresponding genes have yet to be identified in any bacterium, the study of WTA biosynthetic enzymes has been greatly
the GP12 protein produced by the B. subtilis bacteriophage ␸29 enabled by chemoenzymatic syntheses of analogs for the bio-
is thought to possess this activity. GP12 is an appendage protein synthetic intermediates. In place of the 55-carbon undecapre-
found in the neck region of ␸29 that is involved in host cell nyl chain, such analogs feature a more tractable lipid moiety
recognition and entry (25). During phage maturation, GP12 that facilitates manipulation in aqueous systems (8, 30–33).
forms homotrimers that undergo autoproteolysis mediated by Moreover, soluble poly(glycerol phosphate) polymer is accessi-
an intramolecular chaperone domain (26), prior to forming the ble through the polymerization of glycerol phosphate onto
1
2 appendages attached to the neck of phage particles (25, 27). CDP-glycerol (34). Equipped with chemically defined sub-
As part of a crystallographic investigation of this large (92 kDa) strates, we developed HPLC-based assays for poly(glycerol
multifunctional protein, Xiang et al. (27) showed that GP12 phosphate) hydrolysis that were used to meticulously charac-
could drastically reduce the apparent molecular size of poly- terize the wall teichoicase activity of GP12. We found that poly-
(glycerol phosphate) WTA extracted from B. subtilis 168, sug- mer glycosylation led to an enhanced rate of polymer degrada-
1
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Wall Teichoic Acid Degradation by GP12
tion, and also appeared to influence the mode of GP12 action. and at 271 nm for cytidine containing reagents) by reversed
Further interrogation of GP12 catalytic activity suggested that phase HPLC using a Waters HPLC system (Waters 1525 HPLC
the enzyme utilizes phosphodiesterase and phosphomonoes- pump and 2998 PDA detector; Waters, Mississauga, Ontario,
terase activity in its reaction with WTA polymers.
Canada), or by scintillation counting with an in-line 150TR
Flow Scintillation Analyzer (PerkinElmer). Upon reaction com-
pletion, enzymes were removed using a centrifugal filter with a
Experimental Procedures
General Methods—All chemicals were purchased from 10,000 Da molecular mass cut-off (Pall Life Sciences, Missis-
Sigma with the exception of the following: ammonium bicar- sauga, Ontario, Canada).
bonate was from Thermo Fisher Scientific (Waltham, MA),
Synthesis of WTA Analogs—The chemoenzymatic approach
acetonitrile was from Caledon Laboratory Chemicals (George- for the preparation of soluble tricecyl-linked WTA analogs is
town, Ontario, Canada), Tris and urea were from BioShop summarized in Scheme 1. Lipid ␸.40 analog was synthesized
1
4
(
Burlington, Ontario, Canada), sn-[U- C]glycerol 3-phos- using TagF according to established methods, with the lipid ␸.1
14
phate, UDP-D-[U- C]glucose (250 mCi/mmol), and Ultima- analog and CDP-glycerol as the glycerol phosphate acceptor
1
4
Flo M scintillation fluid were from PerkinElmer Life Sciences and donor, respectively (32, 33). To incorporate C into the
14
14
and N-acetyl-D-[U- C]glucosamine (300 mCi/mmol) was glycerol phosphate polymer chain, 0.91 ␮Ci of CDP-[U- C]-
from American Radiolabeled Chemicals Inc. (St. Louis, MO). glycerol (98.7 mCi/mmol) was included in the reaction and the
1
4
The enzymes MnaA, TagA, TagB, TagF, and TagE from B. sub- analogswere prepared at 91 mCi/mmol. [ C]ManNAc lipid
tilis and TarD from S. aureus were purified as previously ␸.40 analog was similarly prepared at 20 mCi/mmol, by the
1
4
14
described (6–8, 32, 35). CDP-glycerol and CDP-[U- C]glyc- inclusion of 0.2 ␮Ci of [ C]ManNAc lipid ␸.1 analog to the
erol (98.7 mCi/mmol) were synthesized using TarD according reaction. Reaction progress was monitored by anion exchange
to established methods (21).
HPLC using a DNA PAC PA200 anion exchange column
Cloning, Expression, and Purification of GP12—The genetic (Thermo Fisher Scientific), with visualization by in-line scintil-
sequence for a GP12 construct missing the first 88 amino acids lation counting. The solvents used were: A, 20 mM ammonium
was PCR-amplified from pET28b-GP12 (27) using primers bicarbonate (pH 8.0) containing 10% acetonitrile (v/v) and B, 20
⌬
N88GP12-Forward and GP12-Reverse (supplemental Table mM ammonium bicarbonate (pH 8.0) containing 1 M NaCl and
S1). The amplicon was cloned into pET28b plasmid using NcoI 10% acetonitrile (v/v). Substrates and products were separated
and HindIII restriction sites to generate p⌬N88GP12. Esche- over the following gradient condition for solvent B: 0–30% (3
richia coli BL21-CodonPlus (DE3)-RIL transformed with min), 30–45% (10 min), 45–100% (5 min), and finally 100% for
p⌬N88GP12 was grown at 25 °C in Luria Bertani (LB) media 5 min. Upon reaction completion, enzymes and low molecular
supplemented with 20 ␮g/ml of chloramphenicol and 50 ␮g/ml weight by-products were removed using centrifugal filters with
of kanamycin to an A of 0.6 before inducing protein expres- molecular mass cut-offs of 30,000 and 3000 Da, respectively
600
sion with 0.1 mM isopropyl ␤-D-1-thiogalactopyranoside. (Pall Life Sciences).
Expression was continued for 20 h at 16 °C. Cells were har- Polymer glycosylation reactions were performed as de-
14
vested by centrifugation (8500 ϫ g for 15 min at 4 °C) and scribed previously (8), by the incubation of TagE with [ C]glyc-
1
4
washed with 0.85% NaCl, prior to resuspension in a buffer con- erol- or [ C]ManNAc lipid ␸.40 analog and a molar excess of
taining Tris-HCl (pH 7.5), 300 mM NaCl, 25 mM imidazole. UDP-glucose to glycerol phosphate units, or likewise with non-
1
4
EDTA-free Protease Inhibitor Mixture (Roche Diagnostics, radioactive lipid ␸.40 analog and UDP-[ C]glucose. The pro-
Laval, Qu e´ bec, Canada) was added to the suspension and the gress of glycosylation reactions was assessed by anion exchange
cells were lysed by two consecutive passages through a cell dis- HPLC using chromatographic conditions identical to those for
ruptor (Constant Systems, Daventry, Northants, UK) at 30,000 the non-glycosylated analog, or by UDP-glucose consumption
p.s.i. Cell lysates were clarified by centrifugation (40,000 ϫ g for with ion-paired reversed phase HPLC (8). Upon reaction com-
6
0 min at 4 °C) prior to loading on a 1-ml HisTrapHP column pletion, enzymes and reaction by-products were removed by
(
GE Life Sciences) pre-equilibrated with resuspension buffer. ultrafiltration as above.
GP12 was eluted over a 25 to 400 mM imidazole gradient, and
Wall Teichoic Acid Degradation Assays—Unless otherwise
fractions containing pure protein were pooled and the buffer stated, GP12 reactions with WTA analogs were performed at
exchanged for 50 mM Tris (pH 7.5), 100 mM NaCl, 15% glycerol room temperature in a buffer comprised of 50 mM Tris-HCl
(
v/v), using a HiPrep 26/10 desalting column (GE Life Sciences). (pH 7.5) and 30 mM MgCl . Reactions were initiated by addition
2
Protein concentration was determined according to the of GP12 and terminated at specified time points by the addition
method of Gill and von Hippel (36). of urea to a final concentration of 6 M. Radioactive reaction
Synthesis of WTA Analog Precursors—Lipid ␣, ␤, and ␸.1 ana- products were separated by anion exchange HPLC as described
1
4
logs were synthesized as described previously (30, 32). above. Initial rate analyses for GP12 degradation of C-labeled
1
4
[
C]ManNAc-labeled lipid ␤ analog (1 mM stock solution; 20 WTA analogs were performed with varying concentrations of
mCi/mmol) was synthesized by including 20 mCi of UDP-N- GP12 and fixed amounts of WTA substrate. The amount of
14
acetyl-D-[1-U- C]glucosamine in the reaction. 1.2 ␮Ci of fully polymeric starting substrate remaining after specific time
1
4
14
[
C]ManNAc lipid ␤ was then used to prepare [ C]ManNAc points was quantified by peak integration.
lipid ␸.1 analog (0.3 mM stock solution; 20 mCi/mmol). Reac-
Liquid Chromatography-Mass Spectrometry—Reactions (30
tion progress was assessed from substrate consumption (mon- ␮l) between GP12 and non-radioactive lipid ␸.40 analogs were
itoring absorbance at 261 nm for uridine containing reagents quenched with an equal volume of methanol, and product mix-
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Wall Teichoic Acid Degradation by GP12
1
4
tures were analyzed by normal phase liquid chromatography
ESI-QTOF mass spectrometry, as described previously (37).
Glycosylation of [ C]glycerol lipid ␸.40 analog using TagE
and UDP-D-glucose likewise yielded a single radioactive prod-
GP12 Hydrolysis of para-Nitrophenyl Phosphates—The cata- uct, as judged by anion exchange HPLC (Fig. 1B). A product
lytic activity of GP12 with pNPP or bis-pNPP was assayed by with an identical retention time was seen on reaction of non-
1
4
monitoring the production of para-nitrophenol (pNP). Unless radiolabeled lipid ␸.40 analog with UDP-D-[ C]glucose, con-
otherwise stated, reactions (100 ␮l) were performed in buffer firming this species as glycosylated polymer (Fig. 1B). MALDI-
containing 50 mM Tris-HCl (pH 7.5), 30 mM MgCl , and 10 mM TOF MS analysis indicated virtually complete modification of
2
CaCl . After initiation with GP12, the absorbance at 400 nm all available glycerol phosphate units with glucose (supplemen-
2
was measured continuously on a SpectraMax microplate reader tal Fig. S1), also reflected by assessment of UDP-glucose con-
(
Molecular Devices, Sunnyvale, CA). pNP generated was quan- sumed in the reaction (not shown).
tified by interpolation to a standard curve.
TagF most efficiently elongates the primer lipid ␸.1 (33), but
Assays for the determination of kinetic parameters con- is also capable of using CDP-glycerol as a primer for poly(glyc-
tained substrate concentrations ranging between 40.0 and erol phosphate) synthesis, forming CMP-poly(glycerol phos-
0
.625 mM for pNPP, and from 28.50 to 0.44 mM for bis-pNPP. phate) (34). We prepared this nucleotide-linked polymer to test
Initial rates of pNP production (substrate consumption Ͻ10%) the generality of GP12 activity and verify that the tridecyl tail
were plotted as a function of substrate concentration and the did not hamper enzymatic degradation. The reaction for
1
4
data fit by non-linear regression (Prism 6.0; GraphPad, La Jolla, [ C]glycerol CMP-poly(glycerol phosphate) WTA showed a
CA) to Equation 1.
single product on anion exchange HPLC analysis (Fig. 1C) and
as previously observed, this analog was estimated to exceed the
polymer length of the lipid ␸.40 analog (8, 34). As with the
tridecyl-linked analog, glycosylation of CMP-poly(glycerol
phosphate) WTA yielded a single product with a shorter reten-
tion time than non-glycosylated (Fig. 1C).
vmax[S]
v ϭ
(Eq. 1)
KM ϩ [S]
Measurement of Inorganic Phosphate—Reactions (50 ␮l) of
GP12 with WTA analogs or bis-pNPP were performed in a
GP12 Catalyzes the Degradation of WTA Analogs—With
buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl and
2
1
4
chemically defined [ C]glycerol-labeled WTA substrates in
hand, we examined the activity of GP12 with these analogs by
monitoring product formation with anion exchange HPLC. Fig.
for bis-pNPP hydrolysis, 5 mM CaCl . At specified time points,
2
reactions were stopped by the addition of EDTA to a final con-
centration of 10 mM, and then 100 ␮l of BIOMOL Green re-
2
, A and B, show the glycerol containing products generated
agent (Enzo Life Sciences, Inc., Farmingdale, NY) was added. P
i
from lipid ␸.40 analogs over time, and it was evident that GP12
catalyzed cleavage of the glycerol phosphate polymer chain.
Whereas products from the reaction with the non-glycosylated
analog spanned a continuum of polymer sizes, a narrow range
was detected by measuring absorbance at 640 nm after 15 min,
and quantified by interpolation to a standard curve.
Results
Characterization of WTA Analogs—Aiming for a polymer of of products corresponding to species of reduced apparent poly-
roughly native length at 40 glycerol phosphate units, we synthe- mer length was produced from reaction of the glycosylated ana-
sized tridecyl-linked WTA analogs using TagF, and CDP- log. These emerged after as little as 2 min and were confirmed to
14
14
[
C]glycerol as the glycerol phosphate donor at 39 times the contain glucose from a parallel reaction of [ C]glucose-glycosy-
molar concentration of lipid ␸.1 acceptor. On completion of the lated lipid ␸.40 analog (Fig. 2B). As well, degradation occurred to a
reaction, anion exchange HPLC analysis showed a single radio- greater extent and with lower GP12 concentrations for the glyco-
active product peak, indicating a polyanionic species formed sylated substrate. Under reaction conditions identical to those for
from the polymerization of glycerol phosphate onto the lipid WTA analogs, we detected no activity for GP12 with glycerol
␸
.1 analog (Fig. 1A). MALDI-TOF analyses of parallel non-ra- phosphate (phosphatase), CDP-glycerol (phosphodiesterase), or
dioactive reactions were consistent with the production of lipid the lipid ␸.1 analog (phosphodiesterase).
.40 analog, but also showed the polymerization product to Similar results were obtained when CMP-poly(glycerol
␸
comprise a mixture of species differing successively by the mass phosphate) WTAs were used as substrates, suggesting no sig-
of the repeating WTA polymer unit (supplemental Fig. S1). nificant influence of the tridecyl tail on activity (Figs. 3, A and
This mass distribution coincides with the previous description B). Subsequent investigations were focused on the tridecyl-
of non-processive polymerization catalyzed by TagF that pre- linked analogs, which could be quantified based on definitive
cludes the generation of a completely homogenous polymer knowledge of the starting concentration of glycerol phosphate
(33). By controlling the ratio of TagF substrates (e.g. lipid ␸.1 acceptor and the polymer size.
analog:CDP-glycerol ratio of 1:39), we were able to target the GP12 More Efficiently Degrades Glycosylated Lipid ␸.40
product mixture to a relatively uniform range of species that Analog—The above results implied GP12 was more active
co-eluted during anion exchange chromatography. The small toward glycosylated WTA analogs. To explore this apparent
differences in polymer sizes (1–5 glycerol phosphate units) preference, we examined activity as a function of time and
contrasts favorably with far greater differences for purified GP12 concentration, monitoring substrate consumption with
WTA or WTA in cell wall extracts (typical polymer sizes of anion exchange HPLC. We note here that substrate concentra-
3
0–50 glycerol phosphate units) and would not adversely affect tions were in large excess of GP12 and substrate consumption
subsequent characterizations of the products from degradation was limited to 15% to ensure measurement of initial rates and to
reactions.
9136 JOURNAL OF BIOLOGICAL CHEMISTRY
favor turnover of the fully polymeric, time 0 substrate. The
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Wall Teichoic Acid Degradation by GP12
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FIGURE 1. Characterization of WTA analogs. Analog structures are shown next to radioactive chromatograms for A, [ C]glycerol lipid ␸.40 analog; B,
1
4
14
glycosylated lipid ␸.40 analog with C incorporated into glycerol (i) and glucose (ii) (prepared by the inclusion of 0.42 mCi of UDP-D-[U- C]glucose (250
mCi/mmol) to a TagE-catalyzed reaction containing non-radiolabeled lipid ␸.40 analog); and C, CMP-poly(glycerol phosphate) WTA analogs. Polymerization of
glycerol phosphate onto CMP-glycerol was accomplished by incubation of CDP-glycerol (3.29 mM) with TagF (34), and glycosylation was performed as with the
1
4
14
14
lipid ␸.40 analog. C was incorporated by the inclusion of either 1 ␮Ci of CDP-[U- C]glycerol (98.7 mCi/mmol) or 0.45 mCi of UDP-D-[U- C]glucose (250
mCi/mmol). Chromatograms with glycosylated CMP-poly(glycerol phosphate) show polymer with C incorporated as [ C]glycerol (i) or [ C]glucose (ii).
1
4
14
14
Under identical chromatographic conditions, retention times for CMP-poly(glycerol phosphate) analogs were slightly increased compared with tridecyl-linked
analogs.
assay showed a linear response with respect to time and enzyme mer degradation, we synthesized WTA analogs labeled with
1
4
concentration and under these conditions, apparent turnover
C exclusively at ManNAc to ensure the detection of only tri-
Ϫ1
by GP12 was estimated at 6.2 and 80 min for non-glycosy- decyl-containing products, as GP12 was not active on the lipid
lated and glycosylated lipid ␸.40 analog, respectively (Fig. 4, A ␸.1 moiety.
and B).
Fig. 5A shows HPLC chromatograms of products from the
Product Distribution Analyses—The degradation of non-gly- reaction of GP12 with lipid ␸.40 analog radioactively labeled as
cosylated and glycosylated analogs seemed to produce different described above. Between 0 and 4 h, the product distribution
1
4
outcomes with respect to the size and distribution of products. resembled that from reactions with the [ C]glycerol-labeled
We hypothesized that this was indicative of different modes of polymer. Further reaction yielded a product distribution
degradation for the two substrates. However, because the start- skewed toward products with smaller apparent polymer sizes,
ing material in either case was labeled throughout the polymer as opposed to the relatively uniform distribution observed for
1
4
chain, products without the tridecyl moiety were indistinguish- [ C]glycerol-labeled products. Hence, the broad variability in
able from those having this appendage. To differentiate product composition from the lipid ␸.40 analog reaction
between these and thereby gain insight into the mode of poly- includes both tridecyl-linked and tridecyl-free species. In con-
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Wall Teichoic Acid Degradation by GP12
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FIGURE 2. GP12 catalyzes cleavage of the glycerol phosphate polymer chain in WTA analogs. Reactions contained 100 ␮M [ C]glycerol-labeled WTA
analog and 1 or 0.1 ␮M GP12, for the non-glycosylated and glycosylated lipid ␸.40 analog, respectively. After termination of the reactions at specified time
1
4
14
points, [ C]glycerol phosphate containing products were separated by anion exchange HPLC. Analog structures, with sites of C incorporation marked by
asterisks, areshownbelowradioactivechromatogramsforA, productsfromthedegradationoflipid␸.40analog;insetshowsthe18-htimepointonamagnified
1
4
scaleandB, productsfromdegradationofglycosylatedlipid␸.40analog;insetshowstheproductsfromreactionof[ C]glucose-glycosylatedlipid␸.40analog.
trast, tridecyl-linked products, significantly reduced in appar-
LC-MS analyses of WTA degradation products offered addi-
ent polymer size, were detected after 2 min of reaction with tional support for the inferred modes of polymer degradation
1
4
[
C]ManNAc-glycosylated lipid ␸.40 (Fig. 5B) and the product (Table 2, supplemental Fig. S2). First, this analysis confirmed
distribution remained skewed toward smaller species. Consid- that cleavage of the polymer occurred at phosphate esters. Fur-
eration of these findings alongside observations with the equiv- thermore, various glycerol phosphate-containing species were
14
alent [ C]glycerol-labeled analog suggests that these trun- identified from a reaction with the non-glycosylated analog,
cated, tridecyl-linked species were co-produced with 2–5-mers whereas species containing one or two (glycosylated) glycerol
of (glucose) glycerol phosphate in the early stages of glycosy- units were detected from a reaction with glycosylated WTA.
lated lipid ␸.40 analog degradation. Collectively, these results Surprisingly, phosphomonoester products anticipated from
portray distinct modes of GP12 action against glycosylated phosphodiester hydrolysis were largely absent from the species
and non-glycosylated substrates; the former is preferentially detected, which was suggestive of monoesterase activity by
hydrolyzed at regular intervals, whereas hydrolysis occurs at GP12.
random sites throughout the latter. Notably, degradation
GP12 Phosphomonoesterase Activity and the Mechanism of
reactions ultimately generated tridecyl-linked products Hydrolysis—The findings from LC-MS analyses were not fully
containing 2–5 monomer units, irrespective of polymer glyco- representative of WTA degradation products apparent from
sylation. It follows that poly(glycerol phosphate) substrates of the HPLC studies, likely because of low ion abundance and/or
GP12 contain at least 3 monomer units.
ion suppression in these experiments. We therefore sought to
1
9138 JOURNAL OF BIOLOGICAL CHEMISTRY
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Wall Teichoic Acid Degradation by GP12
FIGURE 3. Reactions of GP12 with CMP-poly(glycerol phosphate) WTA analogs. Radioactive chromatograms from the anion exchange HPLC separation of
1
4
the products are shown for A, products from [ C]glycerol CMP-poly(glycerol phosphate) degradation; the broad product peak with a slightly reduced
retention time indicates co-elution of multiple species of similar lengths; and B, products from glycosylated CMP-poly(glycerol phosphate) WTA containing
14
14
[
C]glycerol (i) and [ C]glucose (ii).
1
4
FIGURE 4. GP12 exhibits a preference for glycosylated WTA analogs. Activity was assayed by monitoring the disappearance of full-length [ C]glycerol-
labeled WTA analogs by anion exchange HPLC. Data points are the mean from three independent experiments and error bars are the S.D. Insets show plots of
Ϫ1
the initial rates as a function of GP12 concentration and enzyme turnover, calculated as the slope of the linear regression line, was 6.2 and 80 ␮M min , for
non-glycosylated lipid ␸.40 and glycosylated lipid ␸.40, respectively. A, reactions contained lipid ␸.40 analog at 135 ␮M and GP12 at 0.08 (F), 0.16 (E), 0.32(f),
and 0.64 (ꢀ) ␮M. B, reactions contained glycosylated lipid ␸.40 analog at 138 ␮M and GP12 at 0.02 (F), 0.04 (E), 0.08 (f), and 0.16 (ꢀ) ␮M.
establish whether GP12 was additionally capable of catalyzing observed as major products from these reactions (Fig. 6A), and
monoester hydrolysis by initially examining the products from could have possibly arisen from hydrolysis of the corresponding
14
extensive degradation of [ C]glycerol WTA analogs. Dephos- phosphomonoesters generated in preceding phosphodiester
phorylated WTA monomers (glycerol, glucose-glycerol) were hydrolysis. Alternatively, dephosohorylated WTA monomers
JULY 31, 2015•VOLUME 290•NUMBER 31
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Wall Teichoic Acid Degradation by GP12
FIGURE 5. Degradation of non-glycosylated versus glycosylated lipid ␸.40 analogs produces different distributions of tridecyl-linked products.
1
4
Reactions were performed with 200 ␮M [ C]ManNAc lipid ␸.40 analogs. The top two traces in both panels show the elution profiles for standard mixtures
1
4
containing ( C-labeled) UDP-GlcNAc (i), lipid ␤ analog and lipid ␸.1 analog and UDP-GlcNAc (ii) and the product mixture from TagF-catalyzed polymerization
1
4
reaction containing a 3:1 ratio of CDP-glycerol to the lipid ␸.1 analog. Analog structures with sites of C incorporation marked by asterisks are shown below
radioactive chromatograms for A, products from reactions with lipid ␸.40 analog and 1 ␮M GP12; and B, glycosylated lipid ␸.40 analog and 0.1 ␮M GP12.
TABLE 2
Identification of products from GP12-catalyzed degradation of tridecyl WTA analogs by LC-MS
Reactions containing lipid ␸.40 and glycosylated lipid ␸.40 were quenched after 60 and 5 min, respectively. Extracted ion chromatograms and associated mass spectra are
provided in supplemental Fig. S2.
Ion observed (m/z)
Lipid ␸.40 analog
Species
Ϫ
171.02
245.06
399.07
553.07
707.07
701.16
536.17
613.17
690.27
767.18
͓GroP͔
͓Gro-P-Gro͔
͓Gro-P-Gro-P-Gro͔
Ϫ
Ϫ
Ϫ
͓Gro-P-Gro-P-Gro-P-Gro͔
Ϫ
͓Gro-P-Gro-P-Gro-P-Gro-P-Gro͔
Ϫ2
͓Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro͔
Ϫ2
Ϫ2
Ϫ2
Ϫ2
͓(GroP) -ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane͔
2
3
4
5
͓(GroP) -ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane͔
͓(GroP) -ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane͔
͓(GroP) -ManNAc-␤-(1–4)-GlcNAc-1-P-P-tridecane͔
Glycosylated lipid ␸.40 analog
Ϫ
3
33.04
69.18
͓Gro(Glu)-P͔
Ϫ
5
͓Gro(Glu)-P-Gro(Glu)͔
might be the product of diester hydrolysis, especially as glycerol chiometric relative to the input of phosphate in the form of
phosphate was found not to be a substrate for GP12. To clarify, phosphodiester linkages. This was consistent with the modes of
we queried whether P was generated during GP12 reactions action deduced for GP12, whereby not all available phosphodi-
i
with WTA analogs under conditions commensurate with sig- ester linkages are subject to catalytic hydrolysis. Moreover,
nificant polymer degradation (see Figs. 2 and 5). The results of higher P output for the reaction of the glycosylated analog indi-
i
these experiments, presented in Fig. 6B, show that P release cated the presence of increased amounts of hydrolyzable
i
was concomitant with polymer degradation, but was not stoi- monoesters, as could be expected from the greater extent of
1
9140 JOURNAL OF BIOLOGICAL CHEMISTRY
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Wall Teichoic Acid Degradation by GP12
FIGURE 6. Phosphomonoesterase activity of GP12 during WTA analog degradation. A, radioactive chromatograms for products from overnight (18 h)
1
4
reactions of GP12 with [ C]glycerol lipid ␸.40 analogs, after separation by anion exchange HPLC. Control reactions lacking enzyme showed no evidence of
non-enzymatic degradation. B, time course for P generation from degradation of lipid ␸.40 analogs. Reaction mixtures contained 70 ␮M lipid ␸.40 analog and
i
1
␮M GP12 or 70 ␮M glycosylated lipid ␸.40 analog and 0.25 ␮M GP12. Data points are the mean from two independent experiments. C, summary of the
reactions catalyzed by GP12 with WTA substrates. (i) a glycerophosphoglycerol-type product is released as the alcohol leaving group from GP12 phosphodi-
ester hydrolysis. R ϭ H or [glycerol 3-phosphate] ; R ϭ H or glucose. (ii) GP12 hydrolyzes the phosphomonoester product from preceding phosphodiester
1
n
2
hydrolysis to release Pi.
2
ϩ
degradation observed for this analog. We conclude from these ner that was stimulated by Ca (Fig. 7A). Enzyme turnover was
findings that GP12 exercises phosphomonoesterase activity in greater with the diester-containing substrate and indeed, mark-
situ, on products formed during ongoing WTA hydrolysis.
edly higher k and k /K were observed for bis-pNPP (Table
cat
cat
m
The activity of GP12 against WTA substrates therefore 3). In general, the kinetic parameters reflected poor binding and
appeared to involve two hydrolytic reactions, illustrated in Fig. modest turnover for either substrate, but were nonetheless illus-
6
C. The relative abundance of the products of phosphodiester trative of preferential diesterase activity by GP12. Hence, the hy-
cleavage (i.e. alcohol and phosphate monoester) was not clear drolysis of bis-pNPP by GP12 was predicted to yield equimolar
from these reactions with polyphosphodiester WTA sub- amounts of pNPP and pNP in the initial stages of the reaction
strates, presenting a challenge to ascertaining the extent of when [bis-pNPP] ϾϾ [pNPP]. To determine the end result of
phosphate ester hydrolysis. As a result, we employed bis-pNPP pNPP generated during bis-pNPP hydrolysis, we analyzed the
and pNPP as simple substrates to model the reactions catalyzed reaction products by HPLC and found a greater abundance of
by GP12. On the basis of pNP liberation, GP12 was active on pNP,indicatingsubstantialmonoesteraseactivity,i.e.pNPPhydro-
both molecules in an enzyme-concentration dependent man- lysis (Fig. 7B). The ratio of pNPP:pNP implied that GP12 did not
JULY 31, 2015•VOLUME 290•NUMBER 31
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Wall Teichoic Acid Degradation by GP12
FIGURE 7. Catalytic activity of GP12 with bis-pNPP and pNPP. A, reactions (100 ␮l) were performed with 5 mM bis-pNPP or pNPP and GP12 at the concen-
trations indicated. Left and middle panels show the pNP produced after 30 min. Data points are the mean from three independent experiments; errors bars are
the S.D. Right panel shows that the initial rate of pNP production from bis-pNPP (black circles) and pNPP (open circles) was linear with GP12 concentration. B,
HPLC analysis of the products from GP12 hydrolysis of bis-pNPP. After 30 min, reaction mixtures from A were injected onto a C18 reversed phase column.
Separation was achieved by a 10-min gradient of 2–95% CH CN ϩ 0.1% TFA in ddH O ϩ 0.1% TFA. The product mixture was determined to contain 12.1 nmol
3
2
of pNPP and 15.4 nmol of pNP, based on interpolation to standard curves. C, reactions of GP12 with bis-pNPP were assayed for pNP (black circles) and P (open
i
circles), at the time points indicated. Data points are the mean from three independent experiments; error bars are the S.D. The rates of pNP and P production,
i
determined from the slope of the linear regression line between 30 and 120 min, were 2.57 and 0.13 ␮M/min, respectively.
TABLE 3
ited, in previous investigations, to impure enzyme preparations
and/or uncharacterized WTA substrates. Here, we have initiated
the clarification of this facet of WTA biochemistry by using syn-
thetic WTA analogs to investigate teichoicase activity by the GP12
protein from bacteriophage ␸29. To our knowledge, this repre-
sents the first detailed study of such activity for a known enzyme,
with chemically defined WTA substrates.
Kinetic parameters for GP12 hydrolysis of bis-(para-nitrophenyl)
phosphate and para-nitrophenyl phosphate
Given the large amount of WTA in the B. subtilis cell wall, it
may not be surprising to find teichoicase activity of bacterio-
phage origin. An ability to efficiently degrade the glycosylated
WTA encountered at the extracellular surface undoubtedly
aids in overcoming the impediment to penetration posed by the
cell wall. Still, such activity has only been reported for ␸29,
although WTAs are well known bacteriophage cell-surface
receptors (38). It is conceivable that other bacteriophages har-
bor teichoicase activity and indeed, a BLAST query on GP12
returns a number of putative homologs from Bacillus and
Staphylococcus bacteriophages sharing the intramolecular
chaperone and pectin lyase domains in common with GP12.
Interestingly, these domains are also putatively identified in the
product from the B. subtilis gene of unknown function, yobO.
This gene product lacks significant sequence similarity to hypo-
Kinetic parameters were derived by fitting the average initial rates from three inde-
pendent experiments to Eqn. 1. The S.E. of the fit is reported.
catalyze diester and monoester hydrolysis consecutively, which
would have resulted in immediate conversion of the pNPP pro-
duced. This was verified by monitoring the course of P release
i
during bis-pNPP turnover. P generation was observed at a signif-
i
icantly lower rate than pNP (Fig. 7C), providing strong evidence
for catalysis of non-consecutive hydrolytic reactions by GP12 with
phosphodiester-containing substrates.
Discussion
WTAs make up a substantial proportion of the cell wall in
Gram-positive bacteria, where they adopt diverse functional roles.
In B. subtilis, WTA degradation is thought to be part of the phos-
phate starvation response, but may also be important for cell wall thetical B. subtilis teichoicases such as the phosphate starvation
recycling. An understanding of this process has been hampered by proteins PhoD and GlpQ (39), which invokes the possibility of
the absence of known enzymes possessing this activity, and lim- multiple teichoicases encoded by the B. subtilis genome.
1
9142 JOURNAL OF BIOLOGICAL CHEMISTRY
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Wall Teichoic Acid Degradation by GP12
Whether these proteins exhibit teichoicase activity is the sub- products from complex degradation product mixtures. How-
ject of ongoing work in our laboratory.
ever, considering the starting concentrations of diester linkages
That WTA glycosylation was shown to promote more effi- and extent of GP12-catalyzed depolymerization under the
cient polymer degradation by GP12 also seems to coincide with experimental conditions, diester hydrolysis appeared to greatly
the presumed biological function of this protein. In all likeli- exceed P release. We surmise that GP12 catalyzes non-consec-
i
hood, this was primarily due to the different modes that were utive hydrolytic reactions with WTA substrates.
discerned for the action of GP12 against non-glycosylated and
It is implicit from the GP12 crystal structure that the two
glycosylated WTA. Glycosylation unexpectedly appeared to hydrolytic reactions occur at the same catalytic center (27),
exert an effect on the charge properties of WTA, evidenced by although different amino acid residues may be important for
chromatographic profiles (anion exchange) showing the elu- diesterase and monoesterase activities (52, 53). In support of
tion of glycosylated analogs in much lower NaCl concentra- this, it was observed that as diester hydrolysis slowed during the
tions than non-glycosylated analogs with equivalent phosphate bis-pNPP reaction (due to product inhibition by pNP; not
content. Comparable observations have been reported from shown), the rate of P release was unaffected (see Fig. 7C). Inter-
i
polyacrylamide gel electrophoresis of biological WTAs, where estingly, GP12 did not catalyze P release from glycerol phos-
i
D-alanyl modifications can be ruled out as the cause as they are phate, but was active on pNPP, a molecule that bears some
known to be lost during the WTA extraction process (40). structural resemblance to the ligand co-crystallized with GP12
Therefore, glycosylation may counteract potentially unfavor- (N-cyclohexyl-2-aminoethanesulfonic acid) (27). In view of
able interactions with the highly negatively charged WTA poly- this, the regions flanking the phosphate group appear relevant
mer that could hinder productive associations with GP12. As to the specificity of GP12 hydrolysis. This notion is exemplified
well, degradation of non-glycosylated WTA gave rise to diverse by the aforementioned purple acid phosphatases, for which
products that supplied multiple additional substrates for GP12. hydrolytic activity was shown to be affected by such factors as
Hence, as the reaction progresses, there may be increasing the steric accessibility of the substrate and the pK of the poten-
a
competition for the formation of enzyme complexes with the tial leaving group (51, 54). Our findings suggest that GP12 has
full-length (non-glycosylated) substrate, further contributing an increased propensity toward the hydrolysis of phosphodi-
to a reduced rate of degradation. One can imagine testing this ester bonds flanked by bulky substituents (e.g. glucose-glycerol,
with WTA substrates of different polymer lengths and glyco- nitrophenol), and these considerations could have implications
sylation states, although such substrates are difficult to create for the activity of GP12 in a biological setting, where WTA
with current methods.
substrates possess an unspecified organization of ␣-glucose and
Arguably, the product distribution resulting from degrada- D-alanine modifications.
tion of the glycosylated analog matches the expectations for an
In summary, we have conducted the first detailed biochemi-
enzyme that acts processively on a polymeric substrate (41– cal investigation of a purified WTA degrading enzyme, demon-
4
4). Moreover, the GP12 trimer has the capacity to accommo- strating GP12 to be a glycerophosphodiesterase that exercises
date multiple polymeric substrates (27), a property associated diesterase and monoesterase activities in its degradation of
with processively acting enzymes (45). Even so, taking into WTA polymers. This dual capability makes GP12 exceptional
account that GP12 catalyzes two distinct hydrolytic reactions, it among glycerophosphodiesterases and offers an intriguing
would seem that non-processive polymer degradation is a likely potential paradigm for WTA degrading enzymes by highlight-
mode of action for this enzyme.
ing a mechanism for phosphate liberation from WTA. The
The catalysis of two hydrolytic reactions by GP12 is reminis- influence of glycosylation on the efficiency, specificity, and
cent of certain binuclear metallophosphatases that utilize a modality of polymer degradation was an important discovery
bimetallated active site to catalyze the hydrolysis of both phos- because the dispersal of glycosylation sites and other modifica-
phate ester bonds in a diester substrate. Non-consecutive catal- tions throughout the biological polymer remains unclear. GP12
ysis of these hydrolytic reactions has been shown for the pro- and related enzymes may therefore find utility as tools to inves-
miscuous glycerophosphodiesterase GdpQ from Enterobacter tigate structural modifications to WTAs. We hope that the
aerogenes (46–48), bacteriophage ␭ phosphatase (49), and a insight into the enzymology of WTA degradation provided by
cyclic phosphatase dihydrolase from Eggerthella lenta (50). this current work will aid the characterization of novel
Alternatively, purple acid phosphatases from pig and red kid- teichoicases, and find further application in elucidating the
ney bean (51), as well as CthPnkp from Clostridium thermocel- physiological relevance of this activity.
lum (52), are binuclear metallophosphatases that reportedly
catalyze consecutive phosphate ester hydrolyzes; the second Author Contributions—E. D. B. conceived the study and designed
the experiments with C. L. M. R. G. I. purified recombinant GP12,
synthesized WTA analog precursors and performed preliminary bio-
chemical characterizations. T. A. G. performed LC-MS analyses.
C. L. M. performed all other experiments and wrote the manuscript
with E. D. B.
hydrolytic reaction appears to occur without prior enzyme
release of the monoester product from the first reaction. The
hallmark of this mechanism is the production of P at roughly
i
half the rate of the alcohol products from hydrolysis. Accord-
ingly, the rates of P and pNP production during the reaction of
i
GP12 with bis-pNPP reflect discontinuous release of P . The
i
Acknowledgments—pET28b-GP12 plasmid was the kind gift of
Michael Rossman. We thank the Biointerfaces Institute at McMaster
University for performing MALDI-TOF MS experiments.
distinction between consecutive versus non-consecutive phos-
phate ester hydrolysis with WTA substrates could not be easily
gleaned due to the difficulty in resolving monoester and alcohol
JULY 31, 2015•VOLUME 290•NUMBER 31
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Wall Teichoic Acid Degradation by GP12
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