title: specificity and enzyme kinetics of the quorum ... · pdf file2 n-acyl homoserine...

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Title: Specificity and Enzyme Kinetics of the Quorum-

Quenching AHL-lactonase

Running Title: Specificity and Kinetics of AHL-lactonase5

Authors: Lian-Hui WANG*, Li-Xing WENG*, Yi-Hu Dong*, and Lian-Hui

ZHANG*‡§

Affiliation: *Institute of Molecular and Cell Biology, 30 Medical Drive,10

Singapore 117609; ‡Department of Biological Sciences, The

National University of Singapore, 10 Kent Ridge Crescent,

Singapore 119260

§ Author for correspondence: Lian-Hui Zhang15

Institute of Molecular and Cell Biology

30 Medical Drive

Singapore 117609

Tel: +65 6872 7400

Fax: +65 6779 111720

E-mail: [email protected]

JBC Papers in Press. Published on January 20, 2004 as Manuscript M311194200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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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

to produce corresponding N-acyl homoserines. To characterize the enzyme, the5

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

activity towards all tested AHLs varying in length and nature of the substitution at

the C3 position of acyl chain. The data also indicate that the amide group and the10

ketone at the C1 position of the acyl chain of AHLs could be important structural

features in enzyme-substrate interaction. Surprisingly, although carrying a

“104HxHxDH109” short sequence identical to the zinc-binding motif of several groups

of metallohydrolytic enzymes, AHL-lactonase does not contain nor require zinc or

other metal ions for enzyme activity. Except for the amino acid residue H104 that was15

shown previously not required for catalysis, kinetic study and conformational

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.

We conclude that AHL-lactonase is a highly specific enzyme and the20

“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

as quorum sensing (QS) (1-4). In general, each individual bacterial cell produces a basal5

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-

acylhomoserine lactones (AHLs) is a family of QS signals identified in many Gram-

negative bacteria, in particular, Proteobacteria. Different bacterial species may produce10

different AHLs, which vary in the length and substitution of the acyl chain, but maintain

the same homoserine lactone moiety (1, 3, 4). These structural variations could constitute

the basis of signaling specificity of AHL molecules (5, 6).

AHL-dependent QS system has drawn considerable attention over the last 10 years

as it is involved in the regulation of diverse important biological functions, in particular,15

the virulence gene expression in a range of animal (including human) and plant bacterial

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

bacterial infections. Several anti-QS mechanisms have been identified in recent years.20

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,

21, 22). Plants expressing AHL-lactonase quenched pathogen QS signaling and showed5

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

inactivation of QS systems (18, 25).

AHL-lactonase appears to be a potent enzyme. It works well at physiologically10

relevant concentrations of AHL signals, and hydrolyzes the four tested AHLs (C4-HSL, 3-

oxo-C6-HSL, 3-oxo-C8-HSL, and 3-oxo-C12-HSL) effectively (18). The enzyme contains

a conserved short sequence “104HxHxDH109”, which is identical to the Zn2+-binding motif

of several metallohydrolases (26, 27, 28). Within the sequence, three amino acid residues

H106, D108, H109, plus H169 that is also conserved in the metallohydrolases (17), have15

been proven essential for the AHL-lactonase activity (17, 19). It is not clear whether Zn2+

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

compounds. To probe the enzymatic mechanism, the metal ion composition of the enzyme20

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

Synthesis of AHLs and derivatives - AHLs were synthesized as described (6)5

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

sulphoxide (vol:vol) for 12 hr at room temperature. The solution was neutralized to pH 6.5

with 1 M NaH2PO4 and then was dried under vacuum. These synthetic AHLs and Acyl-10

HSs were purified using silica gel column chromatography and C18 reserve-phase HPLC,

and confirmed structurally by 1H NMR spectroscopy and electrospray ionization mass

spectrometry (ESI-MS). Other reagents were purchased from Sigma and Aldrich unless

otherwise stated.

Purification of AHL-lactonase and its variants - The aiiA gene encoding AHL-15

lactonase (AiiA) and its variants H106S, D108S, H109S, and H169S contained in pGEM-

7Zf(+) vector were amplified respectively by PCR using forward primer 5’-

ATCGGATCCATGACAGTAAAGAAGCTTTATTTCG-3’, and reverse primer 5’-

GTCGAATTCCTCAACAAGATACTCCTAATGATGT-3’ (17). The PCR products were

digested by BamHI and EcoRI and fused in-frame to the glutathione S-transferase (GST)20

gene under the control of the isopropyl �-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 �C in 5-liter LB

medium containing 100 �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

incubated at 28 �C overnight. The cells, harvested by centrifugation and resuspended in5

1�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 � g for 30 min, 4 �C). The

supernatant was added to a glutathione Sepharose 4B affinity column (Pharmacia). The

GST fusion proteins were bound to the affinity matrix and AHL-lactonase and its variants

were separated from GST by digestion with the protease thrombin overnight at room10

temperature. After digestion, the eluates containing AHL-lactonase were combined and the

purity was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE). The purified AHL-lactonase and its variants were stored at –80 �C prior to

use. The enzyme concentration was determined by UV spectrophotometry at 280 nm based

on their corresponding molar extinction coefficients (for example, �AHL-lactonase = 18970 M-15

1.cm-1).

AHL hydrolysis and product analysis - The purified AHL-lactonase (0.4 �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�C for 10 min with gentle shaking. After incubation, the mixture was

immediately analyzed by HPLC and ESI-MS, using the same conditions described20

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

quantified by HPLC or bioassay analysis as described (18). AHL-lactonase activity was5

defined as the hydrolyzed �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 �C in 0.1 M phosphate buffer

ranging from pH 5.0-9.0. To examine the thermal stability of AHL-lactonase, the enzyme

solution in 1�PBS buffer (pH 7.4) was allowed to stand for 2 h at various temperatures,10

then the residual activity was measured as described above.

The effects of various metal ions and divalent metal-chelating reagents on the

enzyme activity were examine by incubating AHL-lactonase solution (3 �M) with different

reagents as indicated in 1�PBS buffer at 22 �C for 30 min. The remaining enzyme activity

was measured under the standard conditions described above.15

Metal ion determination - The metal ion composition of AHL-lactonase were

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

adjusted to maximize the signal for analyte ion using standard solutions.20

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 �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

manufacturer’s recommendations. Baseline was corrected with 1�PBS buffer in the5

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

kinetics, AHL-lactonase was added at a final concentration of 1 �M to AHL solution (0.3 –

20 mM) in 0.1 M phosphate buffer (pH 7.4) with a final volume of 96 �l. The reactions10

were incubated at 22 �C, stopped by adding 24 �l of 10% SDS, and subjected to HPLC

analysis. The residual AHL and its hydrolysis product were quantified by HPLC. All

experiments were performed in triplicate and all velocities were determined at time points

at which no more than 10% of the substrate had been consumed. The Kcat and Km values

were calculated based on Michaelis-Menten equation. The enzyme specificity was15

determined by the same procedure except that the substrate and enzyme concentrations

were fixed at 3 mM and 0.5 �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 �M enzyme and 10-200 �M 3-oxo-C8-HSL were

transferred to Centricon-10 tubes (Amicon, Millipore), and centrifuged at 5000 � g until20

the volume of concentrate was about 60-80 �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

amino acid residues (Gly, Ser) at the N-terminus than the native AiiA, was separated from5

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 �g loaded) should be more than 98.5% as staining

with Coomassie brilliant blue R-250, which can detect as little as 0.1 �g protein, did not

reveal other protein bands (Fig. 2). The SDS-PAGE analysis showed the size of the10

purified AHL-lactonase enzyme is ~28 kDa, which is consistent with the predicted

molecular mass of 28,036 Da (17).

The optimal pH for AHL-lactonase activity was examined using 3-oxo-C8-HSL as

substrate. AHL-lactonase activity enhanced with pH increasing from 6.0 to 8.0, reached the

maximum at pH 8.0, then declined slightly at pH 9.0 (Fig. 3A). The potential interference15

of non-enzymatic pH-dependent lactone hydrolysis was precluded by analysis of the

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

adjusted to 5.0 or below.20

AHL-lactonase exhibited excellent thermal stability at temperature below 37�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�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.

In the range between 6 – 37 oC, the activation energy Ea was calculated to be 52.4 kJ.mol-15

from the slope (Ea/R) of the graph. The enthalpy �H� and entropy �S� of activation were

calculated to be 49.9 KJ.mol-1 and -53.7 J.mol-1.K-1, repectively. The free energy �G� of

activation at 25 �C was calculated to be 65.9 KJ.mol-1. These thermodynamic parameters

were calculated by the following equations: �G� = - RTln(kcath/kBT), �H� = Ea - RT, �S� =

(�H� - �G� )/T, where kB, h, and R are Boltzmann, Plank, and universal gas constants,10

respectively.

Several metal ions including Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Zn2+, and Cd2+, showed

no effect on enzyme activity at 0.2 and 2 mM, respectively (Fig. 3C). On the other hand,

AHL-lactonase was partially inhibited by Cr2+ (72%), Pb2+ (67%), and Fe2+ (48%) at 2

mM, and completely inhibited by Cu2+ and Ag+ at 0.2 mM, possibly due to reaction of15

sulfhydryl groups of the enzyme with Cu2+ and Ag+ (30). The chelating reagents such as

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-

lactonase encoded by the aiiA gene is an acidic protein with isoelectric point at 4.7 (17).20

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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electrostatic potentials at the catalytic center, which is critical for enzyme activity. We

used CD spectrometry to determine the effect of pH on AHL-lactonase conformational

structure. Fig. 4 shows that pH has a drastic effect on the conformational structure of AHL-

lactonase. The asymmetric conformational structure of AHL-lactonase remained

unchanged in pH ranging from 7.0 – 9.0, slightly changed at pH 6.0, but significantly5

changed at pH5.5, and completely lost at pH 5.0. The data are consistent with the pH-

dependent enzyme activity pattern of AHL-lactonase (Fig. 3A).

AHL-lactonase is not a metalloenzyme - Sequence alignment suggested that AHL-

lactonase contains a motif similar to the Zn2+-binding motif of metalloenzymes (17). To

determine whether it is a metalloenzyme, we measured Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Cu2+,10

Zn2+, and Pb2+ metal ion contents in AHL-lactonase by ICP-MS. Surprisingly, no metal ion

was detectable except that a trace mount of Zn2+. The Zn content was about 0.08 mol per

mol protein, inconsistent with the notion that it is a metallohydrolase. Moreover, the Zn-

free AHL-lactonase, generated by treatment with chelating reagent EDTA and dialysis, and

confirmed by ICP-MS analysis, maintained the same level of enzymatic activity as the15

untreated enzyme.

AHL hydrolysis by AHL-lactonase - To compare the enzyme activity of AHL-

lactonase toward different AHLs, we synthesized and tested 10 AHL molecules. These

AHLs differ in acyl chain length and substitution at the C3 position of the acyl chain (Fig.

5). AHL-lactonase showed excellent ability to accommodate these structural differences,20

regardless of which AHL was used as a substrate. More than 80% AHL was degraded by

AHL-lactonase in 1:2500 molar ratio of enzyme to substrate in the first 10 min of reaction

and only one new fraction was found after the enzyme reaction by HPLC analysis. The


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new fraction showed the identical HPLC retention time and molecular mass as the

corresponding N-acyl homoserine (Acyl-HS) (data not shown). The Acyl-HSs had not

been detected in the enzyme-free control, indicating that the non-enzymatic turnover of

AHLs, such as pH-dependent lactonolysis or alkalization (31, 32), was negligible in this

time scale. The AHL lactonolysis by AHL-lactonase led to a sharp decrease (over 1500-5

fold) in biological activity, and several products failed to show any activity even at the

concentration up to 10 mM (Table I), when assayed using an A. tumefaciens tra gene based

reporter strain (33). In all cases, the hydrolyzed N-acyl homoserine could not be

relactonized to form active AHL signals in neutral aqueous solution, even after a prolonged

incubation for one week.10

The substrate specificity of AHL-lactonase - The substrate specificity of AHL-

lactonase was studied by determination of the enzyme activity against a range of AHLs,

non-acyl lactones, and non-cyclic esters. The enzyme and substrate was mixed in 1:6000

molar ratio and incubated for 10 min. Fig. 5 shows that the AHL-lactonase exhibited high

relative activities towards all the tested AHLs. Differences in acyl chain length and15

substitution did not significantly affect the enzyme activity, indicating that AHL-lactonase

has a broad catalytic spectrum towards AHLs. Within a narrow margin, AHL-lactonase

worked better against AHLs without 3-oxo substitution than the substituted derivatives. In

contrast, AHL-lactonase showed only residual activity to non-acyl lactones, including

lactones of different members and substituted lactones (Fig. 5). Noticeably, AHL-lactonase20

showed little activity towards γ-decanolactone, which has a 6-carbon alkane side chain.

Several lactonases were also known to digest p-nitrophenyl acetate (34), which is a non-

cyclic ester substrate. However, AHL-lactonase did not hydrolyze any of the tested non-


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cyclic esters, including ethyl acetate, phenyl acetate, p-nitrophenyl acetate, and �-naphthyl

acetate (Fig. 5).

Kinetic analysis of AHL-lactonase - Hydrolysis kinetics was determined by

plotting velocity versus substrate concentration. The kcat and Km values were calculated by

fitting the data to the Michaelis-Menten equation (Table II). AHL-lactonase showed5

comparable catalytic activity against a range of structurally different AHLs with kcat and

Km values ranging from 20.22 – 37.63 s-1 and 1.43 - 7.51 mM, respectively, at pH 7.4 and

22�C. Within these narrow ranges, the enzyme showed higher affinity (Km), slower

hydrolysis rate (kcat), and stronger catalytic efficiency (kcat/Km) toward the AHLs with

longer acyl side chain than the shorter derivatives. Additionally, the enzyme displayed10

higher kcat and kcat/Km values against the AHLs with fully reduced acyl chain than their

corresponding derivatives containing 3-oxo substitution (Table II).

Kinetics and circular dichroism analysis of AHL-lactonase variants deficient in

enzyme activity - The catalytic mechanism of AHL-lactonase has not yet been

characterized. The previous study showed that AHL-lactonases share a conserved motif15

“106HxDH109~H169” which is similar to the Zinc-binding motif of several metallohydrolases

(17, 27, 28). However, the metal ion content analysis indicates that AHL-lactonase is

unlikely to be a metallohydrolase. To study the roles of these conserved amino acid

residues in enzyme catalysis, we prepared four GST-fusion protein constructs to express

and purify four AHL-lactonase variants (H106S, D108S, H109S, H169S). SDS-PAGE20

analysis showed that the AHL-lactonase variants were similar to AHL-lactonase in protein

expression except that replacement of Asp108 with serine (D108S) resulted in a much


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higher level of expression (Fig. 2). The four AHL-lactonase variants were purified and

separated from GST by digestion with site-specific protease thrombin.

To probe the function of the conserved “106HxDH~H169” motif, we analyzed the

enzyme kinetics of four AHL-lactonase variants. Table III shows that single replacement

of D108 and H109 by serine, respectively, led to complete loss of enzyme activity,5

whereas mutation of H106 and H169 to serine resulted in loss of activity by about 47%.

The data are consistent with the previous results based on total soluble protein assay (17,

19). The Km value for the variants H106S and H169S were 10.8 and 13.5 mM,

respectively, using 3-oxo-C8-HSL as substrate. Compared to the Km value of AHL-

lactonase (Table II), the results show that the enzymatic affinity was substantially10

decreased after replacement of H106 and H169 with serine. Kinetic analysis also found

that the catalytic efficiency of H106S and H169S, represented by the kcat/Km values, is 9

and 12 times lower than that of the wild type enzyme (Table III), respectively.

CD spectroscopy was used to monitor the potential protein structural changes

caused by serine substitution of the key amino acid residues. The CD spectrum of AHL-15

lactonase showed an intense peak of negative ellipticity at 202 nm with a small shoulder

peak at 222 nm (Fig. 6), indicating a large unordered contribution and a small but

detectable contribution of �-helical structure. In contrast, the CD spectra of the four

variants differed from their wild type enzyme. Their maximum CD absorbance shifted

from 202 nm to about 206 nm and the intensities of the negative peaks at 222 nm were20

increased, indicating that all variants have higher �-helix contents and lower random coil

contents than the wild type AHL-lactonase (Fig. 6, Table III). The data suggest that the

single amino acid substitution of the key residues involved in enzyme activity caused a


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conformational change, resulted in reduction or lose of the enzyme activity. This

conformational change caused by the single amino acid substitution was also reported in

other proteins, such as human apolipoprotein (35). Interestingly, the CD spectra of H106S

and H169S and those of D108S and H109S are similar, respectively, in particular, at the

structural characteristic negative bands around 206 and 222 nm (Fig. 6). This is in5

agreement with the enzyme activity data that substitution of H106 and H169 respectively

caused a similar level of decrease in enzyme activity, while change either in D108 or H109

has the same detrimental effect (Table III). The data suggest that D108 and H109 might

share a similar functional role, whereas H106 and H169 could be another pair with

equivalent enzymatic contributions.10

The substrate binding ability was determined by centrifugal ultrafiltration (36). The

method is based on centrifugal ultrafiltration through a membrane with a molecular mass

cut-off intermediate between that of the ligand and that of the target protein. The amount of

bound ligand is calculated by subtracting the (free) ligand in the ultrafiltrate fraction from

the total ligand added to enzyme solution. D108S and H109S showed no binding ability to15

substrate 3-oxo-C8-HSL, the added AHL was quantitatively recovered in ultrafiltrate. The

binding ability of H106S and H169S could not be determined by this method due to that

their digestion speeds surpassed the time required for completion of ultrafiltration.

DISCUSSION20

AHL-lactonase has been used previously to demonstrate that quenching bacterial

quorum sensing is a promising strategy to prevent and control bacterial infections (17, 18).

The data presented here show that the AHL-lactonase encoded by the aiiA gene from


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Bacillus sp. 240, is a potent enzyme showing strong catalytic activity and remarkable

substrate specificity against different AHL signal molecules (Table II, Fig. 5). The results

also showed that AHL-lactonase is a not a metallohydrolytic enzyme, in spite of containing

an obvious short stretch of sequence that resembles the zinc-binding motif “HxHxDH” of

several groups of metallohydolase family (17, 26, 27, 28).5

To determine the substrate spectrum of AHL-lactonase and the structural features

of AHLs that are important for enzyme-substrate interaction, we synthesized 9 AHL

compounds, and obtained 3-HO-C4-HSL from a commercial source. By quantification of

the residual AHL and its hydrolysis product, we determined the relative enzyme activity of

AHL-lactonase on different AHL derivatives. The data indicate that AHL-lactonase has a10

broad substrate spectrum, and digests efficiently all the 10 AHL compounds used in this

study, regardless of the length and substitution of the acyl chain (Fig. 5). Though, within a

narrow window of variations, the enzyme activity is somewhat affected by the length and

the substitution at the C3 position of the acyl chain of the substrates. The best substrate of

AHL-lactonase is C6-HSL among the reduced AHL molecules, and 3-oxo-C10-HSL out of15

the C3 substituted AHL signals (Fig. 5). The data indicate that the 3-oxo group of AHLs is

not required for enzyme-substrate interaction.

AHL-lactonase appears highly specific to AHLs showing only residual activity

against L-homoserine lactone and non-homoserine lactones (Fig. 5). The enzyme did not

hydrolyze non-cyclic esters. Among the tested non-acyl lactones, it is interesting to notice20

that the enzyme showed slightly higher residue activity toward L-homoserine lactone than

γ-decanolactone that has a 6-carbon alkane chain (Fig. 5). This observation, together with

the finding that AHL-lactonase has high catalytic activity against all the tested AHL


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derivatives, regardless of the substitution status at C3 position, suggests that the amide

group and the ketone at the C1 position of the acyl chain of AHLs could be important for

enzyme-substrate interaction.

AHL-lactonase shows little homology to other proteins and enzymes except the

significant similarities at two small conserved regions with several groups of5

metallohydrolases, including glyoxalase II, metallo-�-lactamase, and arylsulfatase (17, 26,

27, 28). Sequence alignment of AHL-lactonase with these metallohydrolases revealed that

they share a consensus motif “HxHxDH” at the central region. Crystal structure analysis of

the metallo-�-lactamase from Bacillus cereus showed that the first two histidine residues in

the motif “HxHxDH” were involved in zinc binding, and the aspartic residue was involved10

in catalysis (27). The function of the last histidine residue was not assigned. The site-

directed mutagenesis analysis of AHL-lactonase, however, showed that only the aspartic

and the last two histidine residues, but not the first histidine residue, were required for

enzyme activity (17, 19). It is of significant interest to investigate whether AHL-lactonase,

which appears to have an identical “HxHxDH” zinc-binding motif, is a metallohydrolase.15

The results of this study, however, showed that the purified AHL-lactonase did not contain

detectable amount of Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Cu2+, and Pb2+, except trace amount of

Zn2+ ion. Both removal of the residual Zn2+ from AHL-lactonase by ion chelating reagent

EDTA and addition of Zn2+ to the enzyme did not affect enzyme activity. Apparently,

AHL-lactonase does not belong to metallohydrolase family. We are very curious how the20

HxHxDH sequence serves as a zinc-binding site in some enzymes but not in others.

Several groups of lactonases have been reported (37-39). Among those

characterized lactonases, the gluconolactonase of porcine liver and the lactonohydrolase


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from fusarium oxysporum require divalent metal ions for activity and are inhibited by

metal-chelating reagents (40, 41), whereas others such as 4- and 5-pyridocolactonase and

hydroxyglutaric acid lactonase are independent of such ions for activity (37, 38). AHL-

lactonase appears to belong to the latter.

The AHL-lactonase enzyme activity is pH dependent with the optimal pH at 8.0. A5

shape drop in enzyme activity was noticed when pH was shifted from 6.0 to 5.0; the

enzyme was basically non-active at pH 5.0 (Fig. 3A). CD analysis showed that such a pH

change has a drastic effect on the conformational structure of AHL-lactonase. Changing

pH from 6.0 to 5.0 resulted in a completely loss of the asymmetric structure of the enzyme

(Fig. 4). But partial reverse of both the conformational states of the enzyme and its10

biological activity was noticed when the enzyme solution of pH 5.0 was adjusted to pH 8.0

within half an hour (W.L.H., unpublished data). These results indicate that ionization state

of side chains and H-bonding play a vital role in maintenance of the protein conformational

structure. It is worthwhile to note that pH 5.0 – 6.0 is in the range for side-chain ionization

of histidine residues, and several histidine residues (H106, H109, H169) of AHL-lactonase15

were shown essential for enzyme activity (Table III) (17, 19). Serine substitution of these

key histidine residues respectively did cause certain degree of conformational changes in

AHL-lactonase (Fig. 6), but none of the single replacement could cause a total collapse of

the asymmetric conformational structure of the enzyme. It remains highly intriguing how

these key amino acid residues interact with AHLs and contribute to the catalytic function20

of the enzyme.


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27. Carfi, A., Pares, S. Duee, E., Galleni, M., Duez, C., Frere, J. M., and Dideberg, O.

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32. Yates, E. A., Philipp, B., Buckley, C., Atkinson, S., Chhabra, S. R., Sockett, R. E.,10

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Immun. 70, 5635-5646

33. Piper, K. R., Beck von Bodman, S., and Farrand, S. K. (1993) Nature 362, 448-450

34. Hucho, F., and Wallenfels, K. (1972) Biochimica et Biophysica Acta 276, 176-179.

35. Gursky, O. (2001) Biochem. 40, 12178-1218515

36. Menguy, T., Chenevois, S., Guillain, F., le Maire, M., Falson, P., and Champeil, P.

(1998) Anal. Biochem. 264, 141-148

37. Jong, Y.-J., and Snell, E. E. (1986) J Biol. Chem. 261, 15112-15114

38. Mochizuki, K. (2001) Arch. Microbiol. 175, 430-434

39. Kobayashi, M., Shinohara, M., Sakoh, C., Kataoka, M., and Shimizu, S. (1998) Proc.20

Natl. Acad. Sci. USA 95, 12787-12792

40. Roberts, B. D., Bailey, G. D., Buess, C. M., and Carper, W. R. (1978) Biochem.

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41. Shimizu, S., Kataoka, M., Shimizu, K., Hirakata, M., Sakamoto, K., and Yamada, H.

(1992) Eur. J. Biochem. 209, 383-390

5

10

15

20

25

30

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Figure Legends

Fig. 1. Enzymatic degradation of AHL signals by AHL-lactonase and AHL-acylase.

5

Fig. 2. SDS-PAGE analysis of the expression of GST-AiiA and variants and the purity

of recombinant AHL-lactonase. Lane 1, standard molecular mass marker proteins; Lane

2, purified AHL-lactonase (7 �g); Lanes 3-8, crude cell extract of E. coli containing

construct pGEX-2T; pGST-AiiA; pGST-H106S; pGST-H169S; pGST-D108S; and pGST-

H109S. The protein samples were fractionated by 10% SDS-PAGE gel, following by10

staining with Coomassie brilliant blue R-250. Arrow indicates the location of GST-AiiA

and variants.

Fig. 3. Effect of pH (A), temperature (B), and metal ions (C) on AHL-lactonase

activity.15

Fig. 4. Far-UV CD spectra of AHL-lactonase in phosphate buffer of different pH.

Symbol (upwards at 203 nm): pH 9.0 (x), pH 7.0 (□), pH 6.0 (∆), pH 5.5 (○), pH 5.0 (◊).

Fig. 5. Substrate specificity of AHL-lactonase. Incubations were carried out for 10 min20

at 22 �C in 60 �l reaction mixtures containing 3 mM indicated substrate, 0.5 �M AHL-

lactonase, and 0.1 M phosphate buffer, pH 7.4. The residual substrate was quantified by

HPLC. Data are the means of three to five measurements. The activity towards 3-oxo-C8-

HSL (9.06 �mol.min-1.mg-1) was defined as 100%.


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Fig. 6. Far-UV CD spectra of AHL-lactonase and variants. Symbol (upwards at 200

nm): AHL-lactonase (□), H109S (○), D108S (△), H169S (x), and H106S (◊).


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Table I

Biological activity of AHL hydrolysis products

Samples were diluted and the minimum concentration of each Acyl-HS (AHL

hydrolysis product) required to induce formation of visible blue colony on the bioassay

plate was presented. The data were means of at least three repeats.5

AHL hydrolysis products Minimum concentrationfor activity (�M)a

Decreased activity (times)compared with the

corresponding AHL

3-oxo-C4-HS ND /

3-oxo-C6-HS 500 1667

3-oxo-C8-HS 50 2500

3-oxo-C10-HS 300 1500

3-oxo-C12-HS 1000 1667

C4-HS ND /

C6-HS ND /

C8-HS 1000 2000

C10-HS 2000 2000

3-HO-C4-HS ND /

a ND, not activity was detected in the concentration range from 10 – 10,000 �M.


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Table II

Kinetic parameters of AHL-lactonase against AHLsa

AHLs kcat (s-1) Km (mM) kcat/Km (mM-1.s-1)

3-oxo-C4-HSL 28.63 4.07 7.03

3-oxo-C6-HSL 22.68 2.95 7.69

3-oxo-C8-HSL 22.17 2.28 9.72

3-oxo-C10-HSL 20.22 1.43 14.1

3-oxo-C12-HSLb ND ND ND

C4-HSL 37.63 5.11 7.36

C6-HSL 35.67 3.83 9.31

C8-HSL 27.53 2.61 10.5

C10-HSLb ND ND ND

3-HO-C4-HSL 29.30 7.51 3.90

a The data are means from triplicate experiments.

b ND, not determined due to poor solubility of the substrate in phosphate buffer.5


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27

Table III

Relative enzyme activity and the secondary structure contents of AHL-lactonase and

variants

5

Enzymes Activity (%) Secondary structure contents (%)

�-Helix �-Sheet �-Turn Random

AHL-lactonase 100 6.5 48.2 0 45.3

H106S 53.50 16.5 48.7 2.7 32.1

H169S 53.05 14.8 50.8 4.9 29.5

D108S 0 13.3 52.5 0 34.2

H109S 0 13.6 51.1 0 35.3


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Fig. 1

O

O

NH

OR

O

O

H2N

Acyl-HS

AHL

Fatty acid HSL

AHL-lactonase

AHL-acylase

+

+

H2On

nO

NH

OOH

OH

R

n OH

OR


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Fig. 2

kDa

83.0

175

62.0

47.5

32.5

25.0

1 2 3 4 5 6 7 8


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Fig. 3

0

20

40

60

80

100

120

Non

e

Mg2

+

Ca2

+

Mn2

+

Co2

+

Ni2

+

Zn2+

Cd2

+

Cr2

+

Fe2+

Pb2+

Cu2

+

Ag+

Metal ions

AHL-

lact

onas

e ac

tivity

(%)

0

1

2

3

4

5

4 5 6 7 8 9 10pH

AHL-

lact

onas

e ac

tivity

( �m

ol.m

in-1

.mg-1

)

A

C

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

3.1 3.2 3.3 3.4 3.5 3.6

1/T (103/K)

lnk

cat

B(45�C)

(37�C)

(6�C)


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-12000

-8000

-4000

0

4000

8000

190 200 210 220 230 240 250Wavelength (nm)

Mol

ar e

lliptic

ity (d

eg.c

m2 .d

mol

-1)

Fig. 4


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Substrates Structure Relative activ

1. AHLs

3-oxo-C4-HSL 87.5

3-oxo-C6-HSL 91.8

3-oxo-C8-HSL 100

3-oxo-C10-HSL 109.1

3-oxo-C12-HSL 97.2

C4-HSL 110.9

C6-HSL 124.5

C8-HSL 116.9

C10-HSL 112.4

3-HO-C4-HSL 83.6

2. Other lactones

�-Butyrolactone 2.49

�-Valerolactone 2.10

�-Caprolactone 3.81

L-Homoserine lactone 6.72

�-Valerolactone 2.67

�-Decanolactone 1.85

3. EstersEthyl acetate 0

Phenyl acetate 0

p-Nitrophenyl acetate 0

�-Naphthyl acetate 0

O

ONH

OO

O

ONH

OO

O

ONH

OO

O

ONH

OO

O

ONH

OO

O

ONH

O

O

ONH

O

O

ONH

O

O

ONH

O

O

ONH

OOH

OH2N

O

O

O

O

O

O

O

O

O

O

O

Fig. 5


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-12000

-8000

-4000

0

4000

8000

190 200 210 220 230 240 250Wavelength (nm)

Mol

ar e

lliptic

ity (d

eg.c

m2 .d

mol

-1)

Fig. 6


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Lian-Hui Wang, Li-Xing Weng, Yi-Hu Dong and Lian-Hui ZhangSpecificity and enzyme kinetics of the quorum-quenching AHL-lactonase

published online January 20, 2004J. Biol. Chem.

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