Kinetics and specificity of a quorum-quenching AHL lactonase

34 Pages·2004·0.4 MB·English

JBC Papers in Press. Published on January 20, 2004 as Manuscript M311194200
Title: Specificity and Enzyme Kinetics of the Quorum-
Quenching AHL-lactonase
Running Title: Specificity and Kinetics of AHL-lactonase
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Authors: Lian-Hui WANG*, Li-Xing WENG*, Yi-Hu Dong*, and Lian-Hui
ZHANG*‡§
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10 Affiliation: *Institute of Molecular and Cell Biology, 30 Medical Drive, http
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Singapore 117609; ‡Department of Biological Sciences, The w
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National University of Singapore, 10 Kent Ridge Crescent, brg/
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Singapore 119260 es
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15 § Author for correspondence: Lian-Hui Zhang 18
Institute of Molecular and Cell Biology
30 Medical Drive
Singapore 117609
Tel: [phone]
20 Fax: [phone]
E-mail: [email protected]
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Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
N-acyl homoserine lactone (AHL) quorum-sensing signals are the vital
elements of bacterial quorum-sensing systems, which regulate diverse biological
functions including virulence. The AHL-lactonase, a quorum-quenching enzyme
encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond
5 to produce corresponding N-acyl homoserines. To characterize the enzyme, the
recombinant AHL-lactonase and its four variants were purified. Kinetic and
substrate specificity analysis showed that AHL-lactonase had no or little residue
activity to non-acyl lactones and non-cyclic esters, but displayed strong enzyme
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activity towards all tested AHLs varying in length and nature of the substitution at lo
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10 the C3 position of acyl chain. The data also indicate that the amide group and the fro
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ketone at the C1 position of the acyl chain of AHLs could be important structural ://w
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features in enzyme-substrate interaction. Surprisingly, although carrying a .jb
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“104HxHxDH109” short sequence identical to the zinc-binding motif of several groups by/
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of metallohydrolytic enzymes, AHL-lactonase does not contain nor require zinc or t on
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15 other metal ions for enzyme activity. Except for the amino acid residue H104 that was b
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shown previously not required for catalysis, kinetic study and conformational , 20
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analysis using circular dichroism spectrometry showed that substitution of the other
key residues in the motif (H106, D108, H109), as well as H169 with serine,
respectively, caused conformational changes and significant loss of enzyme activity.
20 We conclude that AHL-lactonase is a highly specific enzyme and the
“106HxDH109~H169” of AHL-lactonase represents a novel catalytic motif, which does
not rely on zinc or other metal ions for activity.
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Many host-associated bacteria produce, release and respond to small signal
molecules to monitor their own population density and control the expression of specific
genes in response to change in population density. This type of gene regulation, which
controls diverse biological functions including virulence and biofilm formation, is known
5 as quorum sensing (QS) (1-4). In general, each individual bacterial cell produces a basal
level of QS signals. The signals accumulate to a threshold concentration as the cells
proliferating, and interact with their cognate transcription factors to activate gene
expression. Several groups of QS signals have been identified. Among them, N-
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acylhomoserine lactones (AHLs) is a family of QS signals identified in many Gram- lo
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different AHLs, which vary in the length and substitution of the acyl chain, but maintain ://w
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the same homoserine lactone moiety (1, 3, 4). These structural variations could constitute .jb
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the basis of signaling specificity of AHL molecules (5, 6). y
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AHL-dependent QS system has drawn considerable attention over the last 10 years t on
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15 as it is involved in the regulation of diverse important biological functions, in particular, b
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the virulence gene expression in a range of animal (including human) and plant bacterial , 2
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pathogens, such as Erwinia carotovora and Pseudomonas aerugunosa (7-14). Being a key
attribute that determines virulence gene expression in pathogenic bacteria, AHL signaling
system has been regarded as a promising target for developing novel approaches to control
20 bacterial infections. Several anti-QS mechanisms have been identified in recent years.
AHL antagonists were found to interfere with bacterial QS signaling, by inducing
accelerated degradation of the AHL-dependent transcription factor (15-16). Two groups of
enzymes, i.e., the acyl-homoserine lactonase (AHL-lactonase) and acyl-homoserine lactone
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acylase (AHL-acylase), which degrade AHL by hydrolyzing respectively the lactone bond
and the amide linkage (Fig. 1), were identified from numerous bacterial isolates (17-24).
Genetically modified E. carotovora and P. aeruginosa expressing AHL-lactonase or AHL-
acylase showed decreased production of virulence factors and attenuated virulence (17, 18,
5 21, 22). Plants expressing AHL-lactonase quenched pathogen QS signaling and showed
significantly enhanced resistance to E. carotovora infection (18). These findings highlight
the promising potential to establish a generic ‘quorum-quenching’ approach to control
bacterial infections, that is, to paralyze quorum-sensing of bacterial pathogens through
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inactivation of QS systems (18, 25). lo
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relevant concentrations of AHL signals, and hydrolyzes the four tested AHLs (C4-HSL, 3- ://w
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oxo-C6-HSL, 3-oxo-C8-HSL, and 3-oxo-C12-HSL) effectively (18). The enzyme contains .jb
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a conserved short sequence “104HxHxDH109”, which is identical to the Zn2+-binding motif by/
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of several metallohydrolases (26, 27, 28). Within the sequence, three amino acid residues t on
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15 H106, D108, H109, plus H169 that is also conserved in the metallohydrolases (17), have mb
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been proven essential for the AHL-lactonase activity (17, 19). It is not clear whether Zn2+ , 2
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or other ions are required for the catalytic function of AHL-lactonase. Little is known
about the substrate specificity and enzyme properties. In this study, we have investigated
the catalytic activity of AHL-lactonase against a range of AHL derivatives and related
20 compounds. To probe the enzymatic mechanism, the metal ion composition of the enzyme
and effect of ions on enzyme activity, have also been determined. Furthermore, four AHL-
lactonase variants deficient in enzyme activity have been purified for kinetic assay and
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conformational analysis using circular dichroism spectrometry in the attempt to reveal the
structural features governing substrate-enzyme interaction and catalytic efficiencies.
EXPERIMENTAL PROCEDURES
5 Synthesis of AHLs and derivatives - AHLs were synthesized as described (6)
except that 3-hydroxylbutanoyl L-homoserine lactone (3-HO-C4-HSL) was purchased
from Quorum Sciences Inc., USA. The acyl homoserines (Acyl-HSs) were prepared by
incubating the corresponding acyl homoserine lactones in 1:1 ration of 1M NaOH/dimethyl
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sulphoxide (vol:vol) for 12 hr at room temperature. The solution was neutralized to pH 6.5 lo
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HSs were purified using silica gel column chromatography and C18 reserve-phase HPLC, ://w
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and confirmed structurally by 1H NMR spectroscopy and electrospray ionization mass .jb
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spectrometry (ESI-MS). Other reagents were purchased from Sigma and Aldrich unless y
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otherwise stated. t on
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15 Purification of AHL-lactonase and its variants - The aiiA gene encoding AHL- b
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lactonase (AiiA) and its variants H106S, D108S, H109S, and H169S contained in pGEM- , 2
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7Zf(+) vector were amplified respectively by PCR using forward primer 5’-
ATCGGATCCATGACAGTAAAGAAGCTTTATTTCG-3’, and reverse primer 5’-
GTCGAATTCCTCAACAAGATACTCCTAATGATGT-3’ (17). The PCR products were
20 digested by BamHI and EcoRI and fused in-frame to the glutathione S-transferase (GST)
gene under the control of the isopropyl (cid:1)-D-thiogalactopyranoside (IPTG)-inducible tac
promoter in GST fusion vector pGEX-2T (Amersham Pharmacia). The constructs were
verified by DNA sequencing.
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Escherichia coli containing different constructs were cultured at 30 (cid:1)C in 5-liter LB
medium containing 100 (cid:2)g.ml-1 of ampicillin. The GST-AHL-lactonase fusion protein and
its variants were expressed by addition of IPTG to a final concentration of 0.5 mM after
the optical density of bacterial culture reached 0.4 - 0.5 at 600 nm; the culture was then
5 incubated at 28 (cid:1)C overnight. The cells, harvested by centrifugation and resuspended in
1(cid:3)PBS buffer (pH 7.4), were disrupted twice with a chilled French pressure cell at 2000
psi. Cell debris was removed by centrifugation (11,000 (cid:3) g for 30 min, 4 (cid:1)C). The
supernatant was added to a glutathione Sepharose 4B affinity column (Pharmacia). The D
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GST fusion proteins were bound to the affinity matrix and AHL-lactonase and its variants ad
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temperature. After digestion, the eluates containing AHL-lactonase were combined and the w
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purity was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis .o
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(SDS-PAGE). The purified AHL-lactonase and its variants were stored at –80 (cid:1)C prior to gu
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use. The enzyme concentration was determined by UV spectrophotometry at 280 nm based e
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15 on their corresponding molar extinction coefficients (for example, (cid:4)AHL-lactonase = 18970 M- er 17
, 2
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1.cm-1). 8
AHL hydrolysis and product analysis - The purified AHL-lactonase (0.4 (cid:2)M) was
mixed with AHL (1 mM) in 1 ml of 0.1 M phosphate buffer (pH 8.0) and the mixture was
incubated at 28(cid:1)C for 10 min with gentle shaking. After incubation, the mixture was
20 immediately analyzed by HPLC and ESI-MS, using the same conditions described
previously (18).
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AHL-lactonase enzyme property analysis - The reaction mixtures containing AHL-
lactonase and substrate in 0.1 M phosphate buffer (pH 7.4) were incubated in water baths
at the specified temperatures. Aliquots were taken at various time points and the reaction
was stopped by adding 10% SDS to a final concentration of 2%. The remaining AHL was
5 quantified by HPLC or bioassay analysis as described (18). AHL-lactonase activity was
defined as the hydrolyzed (cid:2)mols of AHL per minute per mg of AHL-lactonase.
The effect of pH on AHL-lactonase activity was determined by the same procedure
except that the hydrolysis mixtures were incubated at 22 (cid:1)C in 0.1 M phosphate buffer
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ranging from pH 5.0-9.0. To examine the thermal stability of AHL-lactonase, the enzyme lo
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then the residual activity was measured as described above. ://w
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The effects of various metal ions and divalent metal-chelating reagents on the c.o
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enzyme activity were examine by incubating AHL-lactonase solution (3 (cid:2)M) with different g
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reagents as indicated in 1(cid:3)PBS buffer at 22 (cid:1)C for 30 min. The remaining enzyme activity D
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Metal ion determination - The metal ion composition of AHL-lactonase were 18
determined on a Perkin Elmer SCIEX ELAN 6100 inductively coupled plasma mass
spectrometry (ICP-MS). Mass discrimination, auxiliary argon and coolant gas flow rates
were controlled automatically by the instrument. The other operating conditions were
20 adjusted to maximize the signal for analyte ion using standard solutions.
Circular dichroism (CD) spectroscopy - Far-UV CD study of AHL-lactonase and
its variants was carried out on a JASCO J-810 spectropolarimeter at 22 (cid:1)C in the
wavelength range between 185 and 260 nm under constant nitrogen flush, using 1-mm
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path length quartz cells. The spectra were derived from an average of 5 scans recorded at
50 nm.min-1, along with a 1-s time constant. Each spectrum was corrected against blank,
smoothed and analyzed using the software package provided by JASCO. The instrument
was regularly calibrated using ammonium D-(+)-10-camphorsulfonate following the
5 manufacturer’s recommendations. Baseline was corrected with 1(cid:3)PBS buffer in the
absence of enzyme. The fraction of secondary structure was estimated using the method
described previously (29).
Enzyme kinetics and specificity of AHL-lactonase - To determine enzyme
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kinetics, AHL-lactonase was added at a final concentration of 1 (cid:2)M to AHL solution (0.3 – lo
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10 20 mM) in 0.1 M phosphate buffer (pH 7.4) with a final volume of 96 (cid:2)l. The reactions from
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were incubated at 22 (cid:1)C, stopped by adding 24 (cid:2)l of 10% SDS, and subjected to HPLC ://w
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analysis. The residual AHL and its hydrolysis product were quantified by HPLC. All .o
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experiments were performed in triplicate and all velocities were determined at time points gu
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at which no more than 10% of the substrate had been consumed. The K and K values D
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determined by the same procedure except that the substrate and enzyme concentrations 1
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were fixed at 3 mM and 0.5 (cid:2)M, respectively, and the reaction time was 10 min.
Substrate binding assay - To determine the substrate binding ability of the
enzymes, 2 ml solutions containing 20 (cid:2)M enzyme and 10-200 (cid:2)M 3-oxo-C8-HSL were
20 transferred to Centricon-10 tubes (Amicon, Millipore), and centrifuged at 5000 (cid:3) g until
the volume of concentrate was about 60-80 (cid:2)l. The concentrations of 3-oxo-C8-HSL in the
final concentrate and in the filtrate were quantified separately by bioassay analysis.
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RESULTS
Purification and properties of AHL-lactonase - The GST-AiiA fusion protein was
expressed in E. coli following IPTG induction and purified by routine GST affinity
chromatography procedure. The recombinant AiiA (AHL-lactonase), which has two extra
5 amino acid residues (Gly, Ser) at the N-terminus than the native AiiA, was separated from
GST by thrombin digestion. The recombinant enzyme was purified by 86-fold with a yield
about 7.3% of the total proteins. The SDS-PAGE analysis indicates that the purity of the
obtained recombinant AHL-lactonase (7 (cid:2)g loaded) should be more than 98.5% as staining
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with Coomassie brilliant blue R-250, which can detect as little as 0.1 (cid:2)g protein, did not lo
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10 reveal other protein bands (Fig. 2). The SDS-PAGE analysis showed the size of the from
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purified AHL-lactonase enzyme is ~28 kDa, which is consistent with the predicted ://w
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molecular mass of 28,036 Da (17). c
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The optimal pH for AHL-lactonase activity was examined using 3-oxo-C8-HSL as g
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substrate. AHL-lactonase activity enhanced with pH increasing from 6.0 to 8.0, reached the n D
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of non-enzymatic pH-dependent lactone hydrolysis was precluded by analysis of the 0
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controls in which corresponding AHL was incubated in the same reaction buffer without
the enzyme. The enzyme appeared unstable at low pH, which was confirmed by CD
analysis as described in the next section, no or little activity was detected when pH was
20 adjusted to 5.0 or below.
AHL-lactonase exhibited excellent thermal stability at temperature below 37(cid:1)C,
and the purified enzyme kept at 4 oC and 21 oC for 10 days still maintained more than 99%
activity. But the enzyme is less stable at higher temperatures, its activity was decreased
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sharply after incubation for 2 h at above 45(cid:1)C (data not shown). The effect of temperature
on enzyme catalytic activity was analyzed using 3-oxo-C8-HSL as substrate. Up to a
maximum of 37 oC the enzyme activity displayed typical temperature dependence, as
shown in the Arrhenius plot in Fig. 3B, whereas at 45 oC enzyme inactivation was noticed.
5 In the range between 6 – 37 oC, the activation energy E was calculated to be 52.4 kJ.mol-1
a
from the slope (E /R) of the graph. The enthalpy (cid:5)H(cid:1) and entropy (cid:5)S(cid:1) of activation were
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calculated to be 49.9 KJ.mol-1 and -53.7 J.mol-1.K-1, repectively. The free energy (cid:5)G(cid:1) of
activation at 25 (cid:1)C was calculated to be 65.9 KJ.mol-1. These thermodynamic parameters D
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were calculated by the following equations: (cid:5)G(cid:1) = - RTln(kcath/kBT), (cid:5)H(cid:1) = Ea - RT, (cid:5)S(cid:1) = loade
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Several metal ions including Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Zn2+, and Cd2+, showed .org
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no effect on enzyme activity at 0.2 and 2 mM, respectively (Fig. 3C). On the other hand, u
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AHL-lactonase was partially inhibited by Cr2+ (72%), Pb2+ (67%), and Fe2+ (48%) at 2 De
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15 mM, and completely inhibited by Cu2+ and Ag+ at 0.2 mM, possibly due to reaction of er 1
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sulfhydryl groups of the enzyme with Cu2+ and Ag+ (30). The chelating reagents such as 18
EDTA, 2,2’-bipyridine, and o-phenanthroline at a concentration of 2 mM had no effect on
enzyme activity.
The conformational structure of AHL-lactonase is pH-dependent – The AHL-
20 lactonase encoded by the aiiA gene is an acidic protein with isoelectric point at 4.7 (17).
The pH-dependent pattern of the enzyme activity shown in Fig. 3A suggests that the
electrostatic interactions between the charged amino acid residues of AHL-lactonase could
play important roles in maintenance of the overall structural conformation and the local
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