Lipopolysaccharide biosynthesis without the lipids: Recognition promiscuity of Escherichia coli heptosyltransferase i

Article abstract of DOI:10.1021/bi201581b

Heptosyltransferase I (HepI) is responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-d-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k _cat/K_m of 2.7 × 10⁶ M^-1 s ^-1, compared to a value of 2.2 × 10⁵ M^-1 s^-1 for the Kdo₂-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates.

Full text of DOI:10.1021/bi201581b

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Lipopolysaccharide Biosynthesis without the Lipids: Recognition
Promiscuity of Escherichia coli Heptosyltransferase I
Daniel J. Czyzyk, Cassie Liu, and Erika A. Taylor*
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, United States
*
S Supporting Information
between ADP-Hep and the sugar donor domain; however,
ABSTRACT: Heptosyltransferase I (HepI) is responsible
for the transfer of L-glycero-D-manno-heptose to a 3-deoxy-
α-D-oct-2-ulopyranosonic acid (Kdo) of the growing core
region of lipopolysaccharide (LPS). The catalytic efficiency
of HepI with the fully deacylated analogue of Escherichia
coli HepI LipidA is 12-fold greater than with the fully
these structures revealed little about interactions of HepI with
6
the acceptor substrate Kdo -LipidA. Our goal was to identify a
2
substrate analogue that would be amenable to crystallography,
thereby allowing the residues that confer sugar acceptor
specificity to be revealed, while also attempting to identify
tighter binding molecules that lead toward the development of
HepI inhibitors.
Deacylation of the LipidA molecules was investigated to
improve the solubility of the acceptor substrate, thereby
potentially removing the need for detergent, while also
removing potential entropic penalties associated with binding.
6
−1 −1
acylated substrate, with a k /K of 2.7 × 10 M s ,
cat
m
5
−1 −1
compared to a value of 2.2 × 10 M
s
for the Kdo2-
LipidA substrate. Not only is this is the first demonstration
that an LPS biosynthetic enzyme is catalytically enhanced
by the absence of lipids, this result has significant implica-
tions for downstream enzymes that are now thought to
utilize deacylated substrates.
Additionally, because the Kdo -LipidA molecule is believed to
2
be bound in the membrane during catalysis, we hypothesized
that these residues would be relatively unimportant for con-
ferring specificity. Here we report the effect of the acylation
he effectiveness of antibiotics for the treatment of Gram-
state, as well as the number of Kdo molecules, has on K for
m
T
negative infections is hampered by their uptake into cells,
because of the presence of an outer membrane containing a
high level of lipopolysaccharide (LPS). LPS on cell surfaces is
the acceptor substrate. Kdo -LipidA (Figure 1A) was isolated
2
from E. coli HepI knockout strain WBB06 according to
1
7
published protocols, and Kdo-LipidA (Figure 1B, also known
important for cell motility, intestinal colonization and bacterial
biofilm formation, and contributes substantively to antibiotic
as Re-LipidA) was obtained from Sigma-Aldrich. Kdo -LipidA
2
was O-deacylated by reaction with anhydrous hydrazine. The
fully deacylated acceptor molecule (Figure 1D), which was
1
,2
resistance. These characteristics have spurred research toward
the development of inhibitors for the LPS biosynthetic
enzymes. The structure of the LPS is comprised of three
parts: LipidA, core oligosaccharide (Core-OS), and repeating
O-Antigen. Heptosyltransferase I (HepI) catalyzes the first step
in the LPS synthesis pathway following lipid functionalization
4
previously synthesized by Brabetz and coworkers, was
synthesized using a modified method analogous to that used
8
by Bystrova and coworkers. First, the O-deacylated Kdo -
2
LipidA (Figure 1C) was made via reaction with anhydrous
hydrazine and then the fully deacylated acceptor resulted from
subsequent refluxing with 4 M NaOH.
3
,4
of LipidA. Blocking LPS biosynthesis prior to the addition of
an L-glycero-D-manno-heptose (Hep) residue results in increased
bacterial sensitivity to hydrophobic antibiotics and phagocytosis
by macrophages; thus, HepI is considered an excellent target
Using a coupled assay containing pyruvate kinase and L-lactate
dehydrogenase, HepI activity was determined through UV−vis
monitoring of production of nicotinamide adenine dinucleotide
5
6,9
for inhibitor development. In our efforts to determine the
at 340 nm. All four LipidA analogues (Figure 1A−D) were
enzyme mechanism for drug design, we have found that HepI
can efficiently utilize multiple LipidA analogues as substrates,
including a completely lipid free LipidA molecule. This is the
first enzyme in the LPS biosynthetic pathway that does not
found to be competent substrates when the assay was
conducted with or without Triton X-100 (Table 1). For-
mation of the pentasaccharide (Scheme 1) derived from use of
the fully deacylated LipidA analogue (Figure 1D) was con-
firmed by ESI-MS (see the Supporting Information). A roughly
5-fold decrease in the rate of turnover was observed between
the fully acylated acceptor substrates and the deacylated forms
3
have strict selectivity for acylated substrates. Furthermore, this
finding has important implications for the substrate selectivity
of downstream enzymes, which are now thought also to utilize
deacylated substrates.
of Kdo -LipidA, with similar rates observed for both the
2
O-deacylated and fully deacylated LipidA analogues. This decrease
in kcat is more than offset by an increased binding affinity,
causing the overall catalytic efficiency to be 4-fold greater for
HepI is essential for the transfer of the first Hep moiety in
the core oligosaccharide of LPS. It catalyzes formation of an
α(1→5) linkage between L-glycero-D-manno-heptose and the
first 3-deoxy-α-D-oct-2-ulopyranosonic acid (Kdo) covalently
5
attached to LipidA (Scheme 1). A crystal structure of
Received: October 14, 2011
Revised: November 6, 2011
Published: November 7, 2011
Escherichia coli HepI bound to a donor substrate analogue,
ADP-2-deoxy-2-fluoroheptose, reveals binding interactions
©
2011 American Chemical Society
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dx.doi.org/10.1021/bi201581b|Biochemistry 2011, 50, 10570−10572

Biochemistry
Rapid Report
Scheme 1. Reaction Catalyzed by E. coli HepI
the fully deacylated Kdo -LipidA than for the fully acylated
2
6
5
−1 −1
LipidA (k /K values of 2.1 × 10 and 5.2 × 10 M s ,
cat
m
Figure 1. Structures of LipidA acceptor substrates: (A) E. coli Kdo2-
LipidA, (B) Salmonella minnesota Kdo-LipidA, (C) O-deacylated E. coli
Kdo -LipidA, and (D) fully deacylated E. coli Kdo -LipidA. The Kdo
that acts as a nucleophile for the glysyltransferase reaction is colored
red.
respectively) in the presence of Triton X-100 and 12-fold
6
5
−1 −1
greater (k /K values of 2.7 × 10 and 2.2 × 10 M s ,
cat
m
2
2
respectively) in the absence of detergent.
Furthermore, the <2-fold binding affinity discrimination
between Kdo-LipidA and Kdo -LipidA can be rationalized
2
because in vivo it has been demonstrated that the E. coli Kdo
transferase (EcKdtA) is a bifunctional enzyme that catalyzes
incorporation of both Kdo units. Because a single incorporation of
Kdo is only observed using isolated EcKdtA with limiting quantities
in the absence of detergent. We hypothesize that when the acyl
chains are contained within a detergent micelle, they are
isolated from the enzyme, but when free in solution (in the
absence of detergent), they make destabilizing interactions with
the protein surface or aggregate together. This hypothesis is
supported by the observation that HepI aggregates in the
10
of its sugar donor, cytosine monophosphate-Kdo, HepI would
only encounter Kdo -LipidA in vivo, thereby eliminating
2
any driving force for selectivity between Kdo-LipidA and
presence of Triton X-100, the fully acylated Kdo -LipidA, and
2
Kdo -LipidA.
the combination, whereas apo HepI and HepI bound to the
fully deacylated LipidA do not show significant aggregation
when analyzed by size exclusion chromatography (data not
2
Tighter binding of the fully deacylated acceptor analogue,
when compared to the acylated acceptors, is enhanced further
1
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Biochemistry
Rapid Report
Table 1. Kinetic Parameters for the Reaction of HepI with
LipidA Acceptor Molecules in the Presence and Absence of
Detergent
AUTHOR INFORMATION
Corresponding Author
Department of Chemistry, Wesleyan University, Middletown,
■
*
kcat (s−1
k /K (M s−1
−1
CT 06459. Phone: (860) 685-2739. Fax: (860) 685-2211. E-mail:
eataylor@wesleyan.edu.
Funding
D.J.C. thanks the National Institutes of Health Doctoral Studies
in Molecular Biophysics Training Grant 5 T32GM008271 for
support.
acceptor
)
Km (μM)
)
cat
m
A
6.6 ± 0.8
4.7 ± 0.3
8 ± 1
5.5 ± 0.4
1.06 ± 0.05
1.47 ± 0.08
0.76 ± 0.03
1.05 ± 0.07
29 ± 7
9 ± 1
46 ± 13
16 ± 3
0.9 ± 0.2
0.4 ± 0.1
0.28 ± 0.08
0.5 ± 0.1
2.2 × 105
A with Triton X-100
5.2 × 105
1.7 × 105
B
5
B with Triton X-100
3.4 × 10
C
1.1 × 106
3.7 × 106
C with Triton X-100
D
6
ACKNOWLEDGMENTS
We thank Christian Raetz (Duke University, Durham, NC) for
generously providing E. coli HepI knockout strain WBB06.
2.7 × 10
■
D with Triton X-100
2.1 × 106
shown). Furthermore, the observation that the fully deacylated
acceptor has a higher affinity in the absence of detergent, and
the opposite is true for the O-deacylated LipidA analogue,
provides further support for our hypothesis. Because the acyl
portion of the LipidA acceptor resides in the inner leaflet of the
inner membrane when the reaction occurs in vivo, we speculate
that the enzyme only recognizes the sugar region of these
acceptor molecules for catalysis. This is the first report of
enzymatic activity for HepI in the absence of detergent,
suggesting that perhaps the sensitivity of the coupled assay for
determining initial rates is also an improvement over traditional
thin layer chromatography analysis of radioisotope incorpo-
REFERENCES
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11
(
ration for LPS elongation.
The small differences in kinetic parameters among these four
substrates suggest that the sugar binding interactions of the
acceptor substrate predominate for HepI. Our findings are
consistent with a prior report that Kdo -LipidIV (a
(
6
8, 918−925.
(9) Anderson, K. W., and Murphy, A. J. (1983) J. Biol. Chem. 258,
4276−14278.
2
A
1
tetraactylated LipidA analogue that is biosynthetically up-
stream of HepI) can be adventitiously turned over by HepI
(
10) Sirisena, D. M., Brozek, K. A., MacLachlan, P. R., Sanderson,
1
1
K. E., and Raetz, C. R. H. (1992) J. Biol. Chem. 267, 18874−18884.
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and has an apparent K of 4.5 μM. The ability of HepI not
m
(
only to utilize an underacylated LipidA analogue but also to
have improved catalytic efficiency in the absence of any lipids
has broad implications for enzymes that utilize glycolipid
substrates, and especially other enzymes of the LPS
biosynthetic pathway.
2
Furthermore, our finding that fully deacylated Kdo -LipidA is
2
a better substrate for the enzyme than the native substrate, and
that it binds without inducing aggregation, should open up
new strategies for structural analysis of the protein−substrate
interactions of this and related glycolipid-modifying enzymes.
The ability of this enzyme to utilize multiple LipidA analogues
suggests that examination of the glycosyltransferases involved in
LPS biosynthesis, including HepI, could yield answers that are
broadly applicable to questions concerning substrate selectivity
in glycosyltransferases in general. We anticipate that these
results will ultimately enhance lead discovery in the area of
inhibition of LPS biosynthesis for treatment of Gram-negative
bacterial infections.
ASSOCIATED CONTENT
■
*
S
Supporting Information
LPS deacylation procedure, HepI purification, detailed assay
conditions, and ESI-MS data from reaction of the fully
deacylated Kdo -LipidA substrate with HepI. This material is
2
available free of charge via the Internet at http://pubs.acs.org.
1
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dx.doi.org/10.1021/bi201581b|Biochemistry 2011, 50, 10570−10572