In vitro reconstitution of two essential steps in wall teichoic acid biosynthesis.

Abstract

Wall teichoic acids (WTAs) are anionic polymers that decorate the cell walls of many gram-positive bacteria. These structures are essential for survival or virulence in many organisms, which makes the enzymes involved in their biosynthesis attractive targets for the development of new antibacterial agents. We present a strategy to obtain WTA biosynthetic intermediates that involves a combination of chemical and enzymatic transformations. Using these intermediates, we have reconstituted the first two committed steps in the biosynthetic pathway. This work enables the exploration of WTA-synthesizing enzymes as antibiotic targets.

Full text

Page 1

T
exploration of new antibiotic targets (1 ).
Many antibiotics inhibit the biosynthesis
of peptidoglycan, the cross-linked carbo-
hydrate polymer that comprises the major
structural component of the cell wall and
prevents the cell from bursting under high
internal osmotic pressure. Peptidoglycan is
not the only important cell wall component,
however. The peptidoglycan layers in many
Gram-positive organisms are functionalized
with wall teichoic acids (WTAs ), anionic
polymers that are attached to peptidoglycan
via a phosphodisaccharide core (Figure 1; 2 ).
Although their exact functions are unknown,
wall teichoic acids play important biological
roles. They have been shown to be essen-
tial for survival in Bacillus subtilis (3, 4 ) and
to function as virulence factors that promote
Staphylococcus aureus infections (5, 6 ).
The enzymes involved in WTA synthesis
are, therefore, potential targets for the
development of new antibiotics to treat
Gram-positive bacterial infections. Here we
report the preparation of synthetic substrate
analogues and their use in reconstituting
the activities of TagA and TagB, two essen-
tial enzymes in the pathway for wall teichoic
acid biosynthesis in B. subtilis, which is
the major Gram-positive model organism.
This work verifies the proposed functions
of the enzymes and lays the foundation for
detailed mechanistic and structural studies.
WTAs are synthesized as lipid-linked
precursors on the cytoplasmic surface
of the bacterial membrane (Figure 1; 2 ).
he emergence of antibiotic resistance
poses a major threat to human
health, prompting interest in the
The first membrane-bound intermediate,
undecaprenyl-diphospho-N-acetylglu-
cosamine (GlcNAc-pp-und ) (Figure 1 ),
which is formed by the enzyme TagO, is
utilized in the synthesis of several different
anionic cell wall polymers in B. subtilis (7 ).
Therefore, the first committed step in the
biosynthesis of the major WTA in B. subtilis
168 involves the transfer of N-acetylman-
nosamine (ManNAc ) from UDP to the C4
hydroxyl of GlcNAc-pp-und to form the
ManNAc-β-(1,4 )-GlcNAc disaccharide core
of WTA (Figure 1 ). This disaccharide core
is elaborated to a charged polymer by a
series of enzymatic modifications that are
proposed to begin with the addition of a
glycerol phosphate ester to the C4 position
of the ManNAc sugar. The enzymes pro-
posed to catalyze these first two committed
steps in teichoic acid biosynthesis, TagA
and TagB, were identified based on data
from transcriptional fusions (8 ), thermo-
sensitive mutations (9, 10 ), and sequence
homology (11 ). However, the putative
functions of TagA and TagB have not been
demonstrated in vitro because the lipid-
anchored substrates needed to monitor
activity cannot be isolated in useful quanti-
ties from bacterial cells. Moreover, these
substrates contain a 55-carbon ‘carrier’
lipid that is expected to hamper both sub-
strate isolation and kinetic investigations.
Problems related to substrate availabil-
ity and lipid chain length can potentially
be addressed by synthesizing alternative
substrates containing shorter lipid chains.
However, chemical synthesis cannot pro-
vide ready access to the substrates down-
AbstrAct Wall teichoic acids (WTAs ) are
anionic polymers that decorate the cell walls of
many Gram-positive bacteria. These structures
are essential for survival or virulence in many
organisms, which makes the enzymes involved
in their biosynthesis attractive targets for the
development of new antibacterial agents. We
present a strategy to obtain WTA biosynthetic
intermediates that involves a combination of
chemical and enzymatic transformations. Using
these intermediates, we have reconstituted the
first two committed steps in the biosynthetic
pathway. This work enables the exploration of
WTA-synthesizing enzymes as antibiotic targets.
*To whom correspondence should
be addressed.
E-mail: suzanne_walker@
hms.harvard.edu.
Received for review October 2, 2005
and accepted October 28, 2005
Published online January 24, 2006
10.1021/cb0500041 ccc$33.50
Copyright © 2006 by American Chemical Society
In Vitro Reconstitution of Two Essential
Steps in Wall Teichoic Acid Biosynthesis
Cynthia Ginsberg, Yu-Hui Zhang, Yanqiu Yuan, and Suzanne Walker*
Department of Microbiology and Molecular Genetics, Harvard Medical School, and Department of
chemistry & chemical Biology, Harvard University, Boston, Massachusetts 02115
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Page 2

stream of TagA, which contain β linkages to
N-acetyl mannosamine. 1,2-cis-O-Glycosidic
linkages to mannosamine are notori-
ously challenging to construct, and the
ManNAc-β-(1,4 )-GlcNAc linkage found in
WTA has never been made chemically.
Furthermore, synthetic routes to related
compounds are lengthy and involve cum-
bersome functional group interconversions
(12, 13; see Supporting Information. )
Therefore, we decided to explore a chemo-
enzymatic strategy in which TagA itself is
used to make the ManNAc-β-(1,4 )-GlcNAc
linkage found in all downstream substrates
in the WTA pathway.
The natural TagA acceptor contains
a 55-carbon undecaprenyl chain that is
embedded in the bacterial membrane.
Sequence analysis suggests that TagA
does not contain any membrane spanning
regions, and we surmised that the undeca-
prenyl chain is not specifically recognized
by TagA. To simplify the synthesis and
subsequent kinetic studies, we prepared an
acceptor analogue containing a 13-carbon
saturated lipid chain (Scheme 1, 6; 14–17 ).
Following a known route, we also prepared
the required UDP-ManNAc donor sugar 3,
which is not commercially available
(Scheme 1; 18, 19 ).
The ability of TagA to utilize acceptor
analogue 6 was evaluated by incubat-
ing the recombinant enzyme with syn-
thetic substrates 3 and 6 overnight and
analyzing the reaction mixture by liquid
chromatography/mass spectrometry
(LC/MS ). A large peak at m/z = 765 was
observed, consistent with the formation of
the ManNAc-GlcNAc-pp-lipid product. To
assess the efficiency with which TagA uses
6, we measured the reaction rate using
a continuous coupled enzyme assay that
links the production of UDP to the oxida-
tion of NADH (15, 17, 20, 21 ). The apparent
Km of 6 at a UDP-ManNAc concentration of
1.5 mM was found to be 190 ± 30 µM, and
the turnover number was calculated to be
410 ± 50 min–1, consistent with the role
of this glycosyltransferase in the biosyn-
thesis of a primary metabolite. The kinetic
measurements confirm the proposed func-
tion of TagA as a ManNAc transferase and
demonstrate that this synthetic acceptor
analogue is a reasonable substrate to use
in further characterization of TagA.
The high turnover number of TagA
suggested that it would have utility for
preparing the substrate for the next enzyme
in the pathway, TagB. Unlike TagA, TagB has
previously been overexpressed and efforts
to reconstitute activity have been reported
(22 ). Because the disaccharide acceptor of
TagB was not available, however, activity
was monitored by following the incorpora-
tion of radioactivity from CDP-[32P]-glycerol
into bacterial membranes thought to con-
tain the TagB substrate. The reported turn-
over number, 0.004 min–1, is far too low
to keep pace with cell wall biosynthesis,
indicating a problem with either the assay
conditions or the enzyme itself. To obtain
pure substrate to monitor TagB activity,
we converted 2 mg of compound 3 to
compound 7 using TagA and UDP-ManNAc
(Scheme 1 ). In the presence of alkaline
phosphatase to consume UDP, quantitative
conversion to product was achieved within
2.5 h. The identity of the product was con-
O
O
AcHN
O
O
O
HO
O
AcHN
O
O
AcHN
O
HO
O
HO
AcHN
HO
O
L-Ala
γ-D-Glu
m-DAP
D-Ala
D-Ala
O
L-Ala
γ-D-Glu
m-DAP
D-Ala
D-Ala
HO
P
O
-O
O
O
HO
O
AcHN
P
O
O-
O
OR
H
O
HO
NHAc
O
HO
HO
n
P
O
O-
OP
O
O-
P
O
O-
O
P
O
O-
TagA
UDP-
ManNAc
UDP
P
O
O-
O
OH
H
HO
P
O
O-
O
P
O
O-
O
O
O
GlcNAc-PP-
undecaprenyl
ManNAc-GlcNAc-
pp-undecaprenyl
Gro-p-ManNAc-GlcNAc-
pp-undecaprenyl
O
HO
HO
HO
O
NHAc
O
HO
O
HO
O
NHAc
O
HO
AcHN
HO
HO
O
HO
O
HO
O
NHAc
O
HO
AcHN
HO
O
PO
O-
O
P
OO-
CDP-
Glycerol
CMP
O
TagF
Poly-Gro-p-ManNAc-GlcNAc-
pp-undecaprenyl
O
O
HO
O
NHAc
P
O
O-
O
OH
H
O
HO
AcHN
HO
O
P
O
O-
O
OR
H
HO
O
TagGH
P
O
O-
O
P
O O-
O
O
O
HO
O
AcHN
P
O
O-
O
OR
H
O
HO
NHAc
O
HO
HO
?
P
-O
O-
O
TagO
UDP-
GlcNAc
UDP
O
n
UDP-
Glucose
UDP
TagE
R = glucose/ H
n
Peptidoglycan
Glycerol-3-P
CTP
+
TagD
CDP-
Glycerol
CMP
TagB
Figure 1. Polyglycerol phosphate WTA biosynthesis in B. subtilis 168.
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firmed by NMR, with TOCSY spectra used
to make resonance assignments and the
expected β stereochemistry of the linkage
confirmed by large ROESY cross peaks
between H1, H3, and H5 of the ManNAc
sugar (See Supporting Information).
Because the chemical synthesis of 3 and
its subsequent enzymatic transforma-
tion to 7 are straightforward, it should be
possible to obtain more material simply
by scaling up the enzymatic reaction;
however, we note that milligram quantities
of substrates are typically sufficient for
thousands of kinetic assays.
The purified recombinant enzyme was
incubated with substrate 7 and com-
mercially available CDP-glycerol, and
the reaction mixture was analyzed by
LC/MS. After a 3 h incubation, the starting
material 7 was virtually gone and new
peaks were observed at m/z = 459 and
919, consistent with the formation of the
Gro-p-ManNAc-β-(1,4 )-GlcNAc-pp-lipid
product 8 (Scheme 1 ). The appearance
of this product was found to depend on
the presence of active TagB and both
substrates, thus verifying the proposed
function of the enzyme.
The activity of TagB was evaluated
by separating quenched reactions on a
high-performance liquid chromatography
column and quantitating CMP formation
by UV absorbance. The reaction rate was
found to be 1 µM product min-1 nM-1
enzyme, providing an estimated turnover
number of 1000 min–1 under the reaction
conditions. This turnover number is more
than 5 orders of magnitude greater than
that reported previously, highlighting the
advantages of using discrete synthetic
substrates rather than crude bacterial
membrane preparations in studying WTA
synthesizing enzymes. Since both TagA
and TagB are functional in the absence of
detergents and utilize substrates contain-
ing short lipid chains, we conclude that
neither enzyme requires a membrane inter-
face for activity even though their natural
substrates are membrane anchored.
TagA and TagB are potential antibiotic
targets, but they are also of interest for
other reasons. For example, TagA catalyzes
a transformation—attachment of a sugar to
a lipid-anchored monosaccharide—which
is analogous to that performed by the
glycosyltransferase MurG in the biosynthe-
sis of bacterial peptidoglycan (23–24 ) and
also by the second enzyme in the dolichol
pathway for N-linked glycosylation (25 ).
Sequence alignments suggest that TagA is
unrelated to these or other glycosyltrans-
ferases (Gtfs ) for which structural informa-
tion exists, and TagA may thus represent
an uncharacterized Gtf superfamily. TagB
belongs to a class of glycerophosphotrans-
ferases that contains both processive and
nonprocessive enzymes. There is no de-
tailed structural or mechanistic information
for any member of this class of enzymes,
and information on TagB may shed light
on the broader family. Furthermore, we
anticipate that TagB can be used to make
acceptor substrates for TagF, an unusual
processive glycerophosphotransferase
belonging the same enzyme superfamily
as TagB (26, 27 ). Detailed structural and
mechanistic studies of TagA and TagB are
now underway.
METHODS
General Methods. Chemicals and solvents
were from Sigma-Aldrich. Silica gel (60 Å,
32–63 µm ) was from Sorbent Technologies. NMR
spectra were recorded on Varian Inova 400 or
500 MHz spectrometers. Mass spectra (ESI ) were
recorded using an Agilent 1100 series LC/MSD
instrument with an electrospray ionization (ESI )
source in negative ion mode. LC/MS analysis of
enzymatic reactions was also performed on this
instrument, using a Zorbax 300-SB-C18 column
for LC separation.
Synthesis of Compound 3. The triethylammo-
nium salt of 2 (Scheme 1; 0.15 g, 0.239 mmol ),
prepared following published methods (18, 19 ),
and uridine 5´-monophosphomorpholidate
(0.534 g, 0.774 mmol ) were dissolved in 6 mL of
dry pyridine. 1H-Tetrazole (0.050 g, 0.717 mmol )
was added, the reaction was stirred for 48 h at
room temperature (RT ), the solvent was removed,
and the residue was purified over a C-18 column
(0–10% EtOH in 0.1% aqueous NH4HCO3 ) to
give the peracylated precursor of 3. (Spectral
details are provided in Supporting Informa-
tion. ) This compound (0.06 g, 0.077 mmol ) was
dissolved in 4 mL of dry MeOH, and 0.46 mL of
NaOMe (0.228 mmol ) was added. The reaction
was stirred for 1 h at RT and quenched with
CH3COONH4 (1 mL, 0.5 M in H2O ), the solvent
was removed, and the residue was purified over
a C-18 column (0–10% EtOH in 0.1% aqueous
NH4HCO3 ) to give 3 in 30% total yield from 8.
Synthesis of Compound 6. The peracylated
precursor of 6 was prepared following a published
route (Scheme 1; 14-17 ). This compound was
dissolved in 6 mL of dry MeOH, and 1.4 mL of
NaOMe (0.705 mmol ) was added. After 1 h at
RT, the reaction was quenched with CH3COONH4
(2.8 mL, 0.5 M in H2O ), the solvent was removed,
and the residue was purified over a C-18 column
using a gradient of 0–45% EtOH in 0.1% aqueous
NH4HCO3 to give 6 in 79% yield.
Preparation of Compound 7. To a solution of
TagA (1.5 mL, 1 mg mL-1 ) in 20 mM Tris buffer
(pH 7.9, 0.5 M NaCl, 200 mM imidazole ) was
added compound 6 (1.5 mg, 2.5 mmol ), UDP-
ManNAc (3.3 mg, 5.0 mmol ), and 40 µL of alkaline
phosphatase (20 U µL-1 ). After 2.5 h at RT, the
reaction was purified over a C-18 column 0–50%
EtOH in 0.1% aqueous NH4HCO3 to give 7.
Cloning, Expression, and Purification of TagA.
The tagA gene was amplified from Bacillus subti-
lis strain PY79 genomic DNA using two rounds of
PCR. Primers 5´-TGATTTTGCTTTTAGAAACTCTCG-3´
Scheme 1. Preparation of substrates for TagA and TagB.
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ACS CHEMICAL BIOLOGY • VOL.1 NO.1
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O
HO
NHAc
OH
HO
HO
O
AcO
NHAc
AcO
AcO
O P
O
O-
O-
O
HO
NHAc
HO
HO
O P
O
O-
P
O
O-
O
ON
NH
O
O
HO
OH
O
213
O
AcO OAc
AcO
AcO
AcHN
O
AcO
AcO
AcO
O P
O
O-
O-
AcHN
HO
HO
O P
O
O-
P
O
O-
O
O
C13H27
AcHN
645
O
HO
HO
O
O P
O
O-
P
O
O-
O
O
AcHN
7
O
HO
NHAc
HO
HO
TagA
C13H27
100%
P
O
O-
O
OH
HO
CDP-Glycerol
TagB
O
HO
8
H
O
HO
HO
O
O P
O
O-
P
O
O-
O
O
AcHN
O
HO
NHAc
O
HO
C13H27
1000 min-1
i) Ac2O, DMAP, 90%
ii) CF3SO3H, CH2Cl2, 73%
iii) HOPO(OBn)2, toluene
iv) Pd/C, H2, MeOH, Et3N
i) 1H-tetrazole, pyridine,
UMP-morpholidate
ii) NaOMe, MeOH
30% over four steps
i) BnNH2, THF, 78%
ii) iPr2NP(OBn)2, 1H-tetrazole, CH2Cl2
iii) mCPBA, -40oC -> r.t., THF
67% over two steps
iv) Pd/C, H2, MeOH, Et3N
i) CDI, THF
ii) C13H27OPO32-•2Et3N+,
DMF/ THF
68% over three steps
iii) NaOMe, MeOH, 79%
pH 7.5 buffer (20 mM Tris, 100
mM NaCl, 5 mM MgCl2), RT
pH 7.9 buffer (20mM Tris, 0.5M
NaCl, 200mM imidazole), RT, 2.5 h
alkaline phosphatase

Page 4

and 5´-TTTCAGGTTTCACCCATTGC-3´ were
used to amplify a fragment containing tagA.
The gene was then amplified using primers
5´-GTGAGAATTCGATGCAAACAGAGACTATTCAC-3´
and 5´-GTGACTCGAGAATCTGTTTTGTATGATCTTTTTC-3´
and cloned into the EcoRI and XhoI sites
(underlined ) of pET-24b(+ ) (Novagen ). TagA was
expressed in mid-log phase cultures of E. coli
strain Rosetta(DE3 )pLysS (Novagen ), induced with
0.5 mM IPTG for 3 h at 37 °C. Cells were lysed
by freeze–thaw and resuspended in 40 mL of
buffer (20 mM Tris-HCl, pH 7.9, 5 mM imidazole,
0.5 M NaCl ) containing 20 µL of Benzonase
(25 U µL-1 ), 40 µL of protease inhibitor cocktail,
and 40 µL of Triton X-100. The clarified lysate
was purified over a Ni2+ affinity column (Novagen
His·Bind resin) yielding 4 mg L-1 TagA.
Cloning, Expression, and Purification of TagB.
The tagB gene was amplified from Bacillus subtilis
PY79 genomic DNA using primers 5´-GAGCAT-
GTCGCTAGCATGAAAATAAGATCACTACTGG-3´
and 5´-CACTGCAGTCTCGAGGCTTATTAAATTTTC-
GATGAAATT-3´. TagB was cloned into the NheI
and XhoI sites pET-24b(+ ) (Novagen ). TagB was
expressed in mid-log phase cultures of the
E. coli strain BL21(DE3 ) (Novagen ), induced with
0.5 mM IPTG for 22 h at 16 °C. Cells were lysed
with Bugbuster (Novagen ) supplemented with
Benzonase (Novagen ), protease inhibitor cocktail,
and lysozyme following the Novagen protocol.
Clarified lysate was purified over a Ni2+ column
(Novagen IDA His.Bin resin) yielding 12 mg L-1 of
TagB.
Continuous Coupled Enzyme Assay for TagA.
This assay couples the production of UDP to the
oxidation of NADH, leading to a drop in NADH fluo-
rescence (15, 17, 20, 21 ). Reactions were carried
out in 96-well plate microplates (Costar 3603 )
in a volume of 100 µL. NADH fluorescence was
monitored at 465 nM using a Perkin-Elmer HTS
7000 Plus BioAssay reader. Reactions contained
buffer (20 mM Tris, pH 7.9, 5 mM MgCl2, 450 mM
NaCl ), 0.4 U µL-1 PK, 0.2 U µL-1 NDPK, 0.5 mM PEP,
0.2 U µL-1 LDH, 0.25 mM NADH, 0.5 mM ATP, and
0.1 mg mL-1 BSA. Reaction mixtures containing
substrates were incubated at RT for 15 min prior
to adding TagA (100 nM ). Kinetic parameters for
TagA with substrate analogue 6 were calculated as
described in the Supporting Information.
HPLC Assay for TagB. TagB (10 nM ) was
incubated at RT with 200 mM 7 and 400 mM
CDP-glycerol in buffer (20 mM Tris, pH 7.5,
100 mM NaCl, and 5 mM MgCl2 ). Reactions were
quenched with an equal volume of cold methanol,
loaded onto an analytical anion exchange HPLC
column (Phenosphere SAX ), and eluted with a
gradient of 0–56% buffer B over 17 min (buffer
A, 5 mM NH4H2PO4 pH 2.8; buffer B, 750 mM
NH4PO4 pH 3.7 ). CMP was monitored at 260 nm.
Acknowledgment: We thank D. Rudner for
providing purified B. subtilis PY79 genomic DNA.
This work was supported in part by NIH Grant
A144854.
Supporting Information Available: Complete
synthetic and spectral data for compounds 3, 6,
and 7, and enzyme assay details. This material is
available free of charge via the Internet.
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