An acyl-SAM analog as an affinity ligand for identifying quorum sensing signal synthases

Article abstract of DOI:10.1039/c4cc03094j

N-Acylhomoserine lactones (AHLs) are quorum sensing signals produced by Gram-negative bacteria. We here report the affinity purification of AHL synthases using beads conjugated with an enzyme inhibitor, which was designed based on the catalytic intermediate acyl-SAM. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03094j

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An acyl-SAM analog as an affinity ligand for
identifying quorum sensing signal synthases†
Cite this: Chem. Commun., 2014,
50, 8586
Kenji Kai,* Hiroki Fujii, Rui Ikenaka, Mitsugu Akagawa and Hideo Hayashi
Received 25th April 2014,
Accepted 15th June 2014
DOI: 10.1039/c4cc03094j
www.rsc.org/chemcomm
N-Acylhomoserine lactones (AHLs) are quorum sensing signals pro-
duced by Gram-negative bacteria. We here report the affinity purifica-
tion of AHL synthases using beads conjugated with an enzyme inhibitor,
which was designed based on the catalytic intermediate acyl-SAM.
Bacteria communicate using chemical signals to sense cell
density and regulate diverse coordinated behaviors by a process
known as quorum sensing (QS).1,2 N-Acylhomoserine lactones
(AHLs), one class of QS signals in Gram-negative bacteria, are
synthesized from the acyl–acyl carrier protein (acyl-ACP) and
S-adenosylmethionine (SAM) by LuxI-type synthases (Fig. 1a).3,4
This catalytic reaction is thought to be proceeded by a two-step
mechanism involving the intermediate acyl-SAM; the transfer
of an acyl group from acyl-ACP to the amino group of SAM and
lactonization of the methionine part, concomitant with the release
of methylthioadenosine (MTA).3,5,6 AHLs differ in the length and
substitution of their respective acyl side chains, which confers them
with signal specificity.1,2,7 In certain strains, AHLs possessing the
phenylpropanoyl moiety have also been utilized as QS signals.8,9
When the cell density increases and the concentration of AHLs
Fig. 1 (a) Mechanism of AHL production by LuxI-type AHL synthases.
(b) Structures of acyl-SAM analogs (1 and 4) and SAM analogs (2 and 3).
reaches a threshold level, AHLs bind to the cognate LuxR-type has been limited to restricted members of Gram-negative
receptors, and these complexes then activate the expression of bacteria.16,17 As described above, the catalytic mechanism of
target genes. In many pathogenic bacteria, this process results in AHL synthases is thought to be conserved among Gram-
pathogenic events such as the formation of biofilms and produc- negative bacteria; therefore, chemical probes that can bind to
tion of virulence factors.1,2 Therefore, QS modulators have potential AHL synthases may lead to the development of a ligand-based
uses in pharmaceutical and agrochemical fields by preventing approach for identifying and isolating the key enzymes. The
microbial infection in hosts.10–12
competitive inhibitors of AHL synthases may be candidates for the
The identification of AHL synthases is a prerequisite for affinity ligands. Chung et al. described an AHL synthase inhibitor,
understanding AHL-based QS systems in Gram-negative bacteria, named J8-C8, from a chemical library of AHL antagonists and
and mostly depends on screening of genome/cDNA libraries or demonstrated that its inhibitory effect against TofI, an AHL
mutants of target bacteria,8,9,13,14 and more recently on genome synthase of the plant pathogenic bacterium Burkholderia glumae,
analyses.15 Although some degenerated primer sets to amplify was synergistic with the reaction byproduct MTA.18 This finding
AHL synthase genes by PCR have been reported, their application demonstrated, using X-ray crystal analysis, the binding mode of
J8-C8 and MTA to the TofI enzyme of the TofI–J8-C8–MTA com-
plex. However, these compounds only exhibited a weak inhibitory
effect on the TofI enzyme. Christensen et al. recently reported a
Graduate School of Life and Environmental Sciences, Osaka Prefecture University,
1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
strong inhibitor, which inhibited BmaI1, an AHL synthase from
E-mail: kai@biochem.osakafu-u.ac.jp
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc03094j Burkholderia mallei, with a Kai pp value of 0.7 mM.19 This is the first
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inhibitor to inhibit AHL synthase with an effective Ki value, intermediate acyl-SAM may be good competitive inhibitors and
and, thus, opens up a new avenue for the chemical inhibition of affinity ligands of AHL synthases.
bacterial QS. However, it is yet to be determined whether this
Based on the above results, we next designed and synthesized
inhibitor is suitable for an affinity ligand for AHL synthases decarboxy analog 4 (Fig. 1b). This compound could simplify the
because the mechanism of inhibition is noncompetitive; therefore, structure of 1, making it easy to prepare the derivatives of the
the possibility of nonspecific interactions between the inhibitor and octanoyl-SAM analog. Analog 4 was able to inhibit the TofI
proteins other than AHL synthases remains. Therefore, in order to reaction by 91% at 100 mM and 43% at 10 mM (Table 1). The Ki
develop a method for the affinity purification of AHL synthase, we value of 4 was determined to be 6.7 ꢀ 0.7 mM (Fig. S2, ESI†),
firstly need to develop a strong and specific competitive inhibitor of which was similar to that of 1. These results indicated that the
the enzyme.
We speculated that acyl-SAM analogs may be good candidates
carboxy group of analog 1 was not needed to inhibit TofI.
We then attempted to improve the TofI inhibitory activity of 4.
for competitive inhibitors of AHL synthases because many examples Based on a structure–activity relationship study of 4 against TofI
of catalytic intermediate analogs were previously reported to be (see ESI†), we confirmed that the C8-acyl chain including the
good inhibitors of target enzymes,20–24 and the intermediate amide and adenosine moieties of 4 were recognized by TofI and,
acyl-SAM was shown to be specific to the synthesis of AHL in thus, suggested that the enzyme may have high affinity to the
Gram-negative bacteria.3,5,6 Furthermore, to the best of our catalytic intermediate octanoyl-SAM. The findings of a previously
knowledge, acyl-SAM has not yet been detected in other organisms; conducted crystallographic analysis indicated that J8-C8 and
thus, the analogs may also be useful for identifying the AHL MTA bound to TofI, occupying the binding sites of the acyl chain
synthase of an uncultured bacterial symbiont from the host cell of octanoyl-ACP and the adenosine moiety of SAM, respectively.18
lysate.25 In the case of TofI, N-octanoylhomoserine lactone (C8-HSL) Therefore, it was reasonable to speculate that the binding mode
was synthesized from octanoyl-ACP and SAM.18 Hence, we firstly of the respective substructures of octanoyl-SAM to the enzyme
designed and synthesized N-octanoyl-S-adenosyl-L-homocysteine (1) was similar to those of J8-C8 and MTA observed in the ternary
(Fig. 1b) as an octanoyl-SAM analog, which lacked the methyl group complex. If this was the case, J8-C8 and MTA in the crystal could
at the 50-sulfur atom of the intermediate. The activity of 1 was be replaced by octanoyl-SAM. However, the crystallographic
tested for its ability to inhibit AHL synthase using octanoyl-ACP analysis also revealed that the binding sites of J8-C8 and MTA
(10 mM), which was prepared from the ACP of Escherichia coli K-12 were separated in TofI and also the presence of the large channel
by chemical acylation,26,27 with SAM (10 mM) as the substrate and in which the reactions for acylation and lactonization of the
the TofI enzyme (1 mM), which was heterogeneously expressed in methionine part in SAM can occur. Thus, the pocket of the TofI
E. coli strain BL21(DE3)pLysS.18 The C8-HSL product was quantified crystal appeared to be slightly larger than the size of octanoyl-
by GC–MS.25,28 Analog 1 showed 91% and 51% inhibition at SAM. Therefore, we predicted that the length between the sulfur
concentrations of 100 and 10 mM, respectively (Table 1). Kinetic atom and amide in octanoyl-SAM analogs may be an important
analysis indicated that 1 acted as a competitive inhibitor of TofI factor and that elongation of the methylene chain of 4 could
activity with a Ki value of 4.8 ꢀ 0.7 mM, which was determined by a improve the inhibitory activity of 4 against TofI.
Dixon plot of 1/V0 versus the inhibitor concentration (Fig. S1, ESI†).
Thus, we designed and synthesized analogs 5–8 (Fig. 2a) in
On the other hand, the SAM analogs, S-adenosylhomocysteine (2) which the methylene chains between the sulfur atom and amide in
and sinefungin (3) (Fig. 1b), showed moderate or no inhibition of 4 were extended to C4–C7, respectively. As expected, the inhibitory
TofI at the concentrations tested (Table 1), although they were activities of these analogs were higher than that of 4, and they
reported to have some inhibitory effects on AHL synthase.3 There- almost completely inhibited the production of C8-HSL by TofI at a
fore, it is reasonable to assume that mimics of the reaction concentration of 10 mM, while 5, 6, and 7 were effective to a similar
degree at 1 mM (Table 1). Analogs 6 and 7 showed high inhibitory
Table 1 TofI inhibition by acyl-SAM analogs
activity against TofI with Ki values of 0.22 ꢀ 0.03 and 0.11 ꢀ 0.02 mM,
respectively (Fig. S3 and S4, ESI†). A docking simulation provided a
Inhibitiona (%)
reasonable model in which analog 6 bound to the pocket of TofI
Compound
100 mM
10 mM
1 mM
Kib (mM)
covering the acyl chain of J8-C8 and the adenosine of MTA (Fig. 2b,
Fig. S6a and b, ESI†). However, in a docking model of 7 in TofI, the
analog did not match the model of 6, rather the acyl chain of 7 fit to
the upper space of the binding site of that of 6 (Fig. S6c, ESI†). The
possibility remains that analog 7 also binds to TofI in the same
manner as that of 6. Future experiments (e.g., X-ray crystallographic
analyses of these inhibitors with TofI) are needed to clarify their
binding mode. Because analog 8 showed lower activity than analogs
5–7, the methylene chains between the sulfur atom and amide in 8
1
2
3
4
5
6
7
8
9
91
51
n.i.
91
51
8
n.i.
43
87
91
498
83
88
4.8
12
40
52
83
21
48
51
6.7
0.73
0.22
0.11
0.88
10
94
a
The percentage inhibition was calculated from the ratio of C8-HSL were too long to fit well into TofI. Analogs 6 and 7 were conclusively
production with and without an inhibitor; n.i.
= no inhibition
found to be effective inhibitors that satisfied the binding site scaffold
of the TofI enzyme. Due to its high hydrophobicity, analog 7
appeared to be difficult to handle during the preparation of
(i.e., o5% inhibition). Data represent the average of triplicate determi-
b
nations. Ki values were determined by a Dixon plot of V0 versus the
inhibitor concentration.
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Fig. 2 (a) Structures of analogs 5–8. (b) A docking model of analog 6 to the
TofI enzyme. Stick model of analog 6: carbons, gray; hydrogens, white; oxygens,
red; nitrogens, blue; sulfur, green. Stick model of J8-C8: yellow, left. Stick model
of MTA: yellow, right. The binding site in TofI is shown as a surface model.
Fig. 4 Pull-down assays of AHL synthases. (a) Purified TofI. (b) Whole cell
protein extracts of E. coli expressing TofI. (c) Whole cell protein extracts of
E. coli expressing BmaI1. (d) Whole cell protein extracts of E. coli expres-
sing YspI. Beads that bound proteins were incubated with buffer containing
6 (LE, ligand elution). The remaining proteins on the beads were liberated
by boiling the beads (BE, boiling elution). Fractions collected were analyzed
by SDS-PAGE (15% acrylamide) followed by silver staining. Inp: whole cell
protein extracts of E. coli.
affinity beads. Therefore, we selected 6 as an affinity ligand for
the isolation of AHL synthases.
The above docking simulation of analog 6 to TofI also suggested
that the 20- and 30-hydroxy groups of analog 6 presented almost at
the surface of the enzyme (Fig. S6b, ESI†). Therefore, we introduced
a propargyl linker to either of the hydroxy groups in an ether form,
and the resulting alkynes 9 and 10 that formed were fixed to azide-
type magnetic beads (FG beads)29 using click chemistry (Fig. 3).
Ligands 9 and 10 also inhibited TofI to the same extent as analog 6
(Table 1). Thus, we confirmed that the propargyl linkers did not
affect their inhibitory activity or binding to TofI. The results of pull-
down assays of the ligand-fixed beads using purified TofI are shown
in Fig. 4a. Control beads (no ligand) did not bind to TofI. In
contrast, TofI was bound to 9- and 10-fixed beads and was effectively
eluted by 6 solution (250 mM). These results indicated that these
ligand-fixed beads possessed the ability to capture the TofI protein.
Thus, 9- and 10-fixed beads were used in pull-down assays with the
whole cell protein extracts of E. coli strain BL21(DE3)pLysS expres-
sing TofI. The results obtained demonstrated that only TofI was
purified from crude samples (Fig. 4b), which indicated that the
interaction between TofI and the ligand-fixed beads was very
specific. We also confirmed the competitive inhibition of their
interactions by the natural ligand SAM (Fig. S7, ESI†). There-
fore, these results suggested that 9- and 10-fixed beads were a
good affinity matrix for AHL synthases.
Finally, to address the utility of 9- and 10-fixed beads in the
purification of other AHL synthases, we carried out pull-down
assays of BmaI1, a Burkholderia mallei AHL synthase, and YspI,
a Yersinia pestis AHL synthase,19 using the beads. As a result of
the pull-down assay of the whole cell protein extracts of E. coli
strain Tuner(DE3) expressing BmaI1, we obtained BmaI1 as a
major band from the samples obtained by ligand and boiling
elutions of ligand-fixed beads (Fig. 4c). The binding of BmaI1 to
the ligand-fixed beads should be stronger than that of Tof I because
BmaI1 was difficult to elute with 6 solution, but was effectively
eluted by boiling the beads in the SDS sample buffer. 10-fixed
beads captured BmaI1 more effectively than 9-fixed beads, suggest-
ing that the preparation of several conjugation-type beads is needed
to successfully isolate AHL synthases by pull-down assays. In
contrast, YspI could not be effectively purified by pull-down
assays from the E. coli cell lysate, in which only weak binding of
YspI to the ligand-fixed beads was observed (Fig. 4d). This was
attributed to YspI mainly producing N-(3-oxooctanoyl)homo-
serine lactone (3-oxo-C8-HSL) and N-(3-oxohexanoyl)homoserine
lactone (3-oxo-C6-HSL),30 while BmaI1 produced C8-HSL.19
Therefore, we confirmed the specificity of these beads for
C8-HSL-type AHL synthases.
Fig. 3 Structures of 9, 10, and 9-fixed beads.
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In conclusion, we developed strong AHL synthase inhibitors,
which were designed based on the catalytic intermediate acyl-
SAM, for affinity ligands of AHL synthases. We also demonstrated
the usefulness of acyl-SAM analog-fixed beads in pull-down assays
to isolate AHL synthases. This is the first study to show that QS
signal synthases could be purified by a ligand-based affinity
protocol. Furthermore, acyl-SAM analogs are expected to be the
lead compounds for antivirulence agents because they showed
high specific binding to AHL synthases. The identification of AHL
synthases and their genes is currently based on gene/mutant
screening or genome analysis. In addition to these approaches,
acyl-SAM analog-based affinity ligands will provide a new method
to identify unknown AHL synthases in Gram-negative bacteria.
We gratefully acknowledge Prof. Ingyu Hwang (Seoul National
University) and Prof. E. Peter Greenberg (University of Washington)
for providing plasmids, Dr Toshiyuki Harada (Sumitomo Chemical)
for his assistance in constructing docking models, Prof. Naoharu
Watanabe and Dr Hiroyuki Takemoto (Shizuoka University) for
HRMS measurements, and Prof. Jun Hiratake (Kyoto University)
for his valuable discussions. This work was supported by JSPS
KAKENHI Grant Number: 23658101, 26450140.
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