Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase)
2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês
10.1074/jbc.m311194200
ISSN1083-351X
AutoresLianhui Wang, Lixing Weng, Yi‐Hu Dong, Lian‐Hui Zhang,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoN-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 quorumquenching enzyme encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond 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 noncyclic esters, but displayed strong enzyme activity toward all tested AHLs, varying in length and nature of the substitution at the C3 position of the acyl chain. The data also indicate that the amide group and the ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction. Surprisingly, although carrying a 104HX- HXDH109 short sequence identical to the zinc-binding motif of several groups of metallohydrolytic enzymes, AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity. Except for the amino acid residue His-104, which was shown previously to not be required for catalysis, kinetic study and conformational analysis using circular dichroism spectrometry showed that substitution of the other key residues in the motif (His-106, Asp-108, and His-109), as well as His-169 with serine, respectively, caused conformational changes and significant loss of enzyme activity. We conclude that AHL-lactonase is a highly specific enzyme and that the 106HXDH109∼H169 of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity. 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 quorumquenching enzyme encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond 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 noncyclic esters, but displayed strong enzyme activity toward all tested AHLs, varying in length and nature of the substitution at the C3 position of the acyl chain. The data also indicate that the amide group and the ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction. Surprisingly, although carrying a 104HX- HXDH109 short sequence identical to the zinc-binding motif of several groups of metallohydrolytic enzymes, AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity. Except for the amino acid residue His-104, which was shown previously to not be required for catalysis, kinetic study and conformational analysis using circular dichroism spectrometry showed that substitution of the other key residues in the motif (His-106, Asp-108, and His-109), as well as His-169 with serine, respectively, caused conformational changes and significant loss of enzyme activity. We conclude that AHL-lactonase is a highly specific enzyme and that the 106HXDH109∼H169 of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity. 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) 1The abbreviations used are: QS, quorum-sensing; AHL, acyl homoserine lactone; Acyl-HS, acyl homoserine; HPLC, high pressure liquid chromatography; GST, glutathione S-transferase; IPTG, isopropyl β-d-thiogalactopyranoside; PBS, phosphate-buffered saline; ICP-MS, inductively coupled plasma mass spectroscopy; HSL, homoserine lactone. (1Fuqua C. Parsek M.R. Greenberg E.P. Ann. Rev. Genet. 2001; 35: 439-468Google Scholar, 2Winans S.C. Trends Microbiol. 1998; 6: 382-383Google Scholar, 3Miller B. Bassler B.L. Annu. Rev. Microbiol. 2001; 55: 165-199Google Scholar, 4Whitehead N.D. Barnard A.M.L. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Google Scholar). In general, each individual bacterial cell produces a basal level of QS signals. The signals accumulate to a threshold concentration as the cells proliferate and interact with their cognate transcription factors to activate gene expression. Several groups of QS signals have been identified. Among them, N-acyl homoserine lactones (AHLs) comprise a family of QS signals identified in many Gram-negative bacteria, in particular, Proteobacteria. Different bacterial species may produce different AHLs, which vary in the length and substitution of the acyl chain but maintain the same homoserine lactone moiety (1Fuqua C. Parsek M.R. Greenberg E.P. Ann. Rev. Genet. 2001; 35: 439-468Google Scholar, 3Miller B. Bassler B.L. Annu. Rev. Microbiol. 2001; 55: 165-199Google Scholar, 4Whitehead N.D. Barnard A.M.L. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Google Scholar). These structural variations could constitute the basis of signaling specificity of AHL molecules (5Welch M. Todd D.E. Whitehead N.A. McGowan S.J. Bycroft B.W. Salmond G.P.C. EMBO J. 2000; 19: 631-641Google Scholar, 6Zhang L.H. Murphy P.J. Kerr A. Tate M.E. Nature. 1993; 362: 446-448Google Scholar). The AHL-dependent QS system has drawn considerable attention over the last 10 years, as it is involved in the regulation of diverse and important biological functions, in particular, the virulence gene expression in a range of animal (including human) and plant bacterial pathogens such as Erwinia carotovora and Pseudomonas aeruginosa (7Jones S. Yu B. Bainton N.J. Birdsall M. Bycroft B.W. Chhabra S.R. Cox A.J.R. Golby P. Reeves P.J. Stephens S. Winson M.K. Salmond G.P.C. Stewart G.S.A.B. Williams P. EMBO J. 1993; 12: 2477-2482Google Scholar, 8Passador L. Cook J.M. Gambello M.J. Rust L. Iglewski B.H. Science. 1993; 260: 1127-1130Google Scholar, 9Pirhonen M. Flego D. Heikinheimo R. Palva E. EMBO J. 1993; 12: 2467-2476Google Scholar, 10Pearson J.P. Gray K.M. Passador L. Tucker K.D. Eberhard A. Iglewski B.H. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 197-201Google Scholar, 11Beck von Bodman S. Farrand S.K. J. Bacteriol. 1995; 177: 5000-5008Google Scholar, 12Costa J.M. Loper J.E. Can. J. Microbiol. 1997; 43: 1164-1171Google Scholar, 13Dunphy G. Miyamoto C. Meighen E. J. Bacteriol. 1997; 179: 5288-5291Google Scholar, 14Nasser W. Bouillant M.L. Salmond G. Reverchon S. Mol. Microbiol. 1998; 29: 1391-1405Google Scholar). Being a key attribute that determines virulence gene expression in pathogenic bacteria, the AHL signaling system has been regarded as a promising target for developing novel approaches to controlling 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 (15Givskov M. de Nys R. Manefield M. Gram L. Maximilien R. Eberl L. Molin S. Steinberg P.D. Kjelleberg S. J. Bacteriol. 1996; 178: 6618-6622Google Scholar, 16Manefield M. Rasmussen T.B. Henzter M. Andersen J.B. Steinberg P. Kjelleberg S. Givskov M. Microbiology. 2002; 148: 1119-1127Google Scholar). Two groups of enzymes, i.e. the acyl-homoserine lactonase (AHL-lactonase) and acyl-homoserine lactone acylase (AHL-acylase), which degrade AHL by hydrolyzing, respectively, the lactone bond and the amide linkage (Fig. 1), were identified from numerous bacterial isolates (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar, 18Dong Y.H. Wang L.H. Xu J.L. Zhang H.B. Zhang X.F. Zhang L.H. Nature. 2001; 411: 813-817Google Scholar, 19Dong Y.H. Gusti A.R. Zhang Q. Xu J.L. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 1754-1759Google Scholar, 20Lee S.J. Park S.Y. Lee J.J. Yum D.Y. Koo B.T. Lee J.K. Appl. Environ. Microbiol. 2002; 68: 3919-3924Google Scholar, 21Reimmann C. Ginet N. Michel L. Keel C. Michaux P. Krishnapillai V. Zala M. Heurlier K. Triandafillu K. Harms H. Defago G. Haas D. Microbiology. 2002; 148: 923-932Google Scholar, 22Lin Y.H. Xu J.L. Hu J. Wang L.H. Ong S.L. Leadbetter J.R. Zhang L.H. Mol. Microbiol. 2003; 47: 849-860Google Scholar, 23Leadbetter J.R. Greenberg E.P. J. Bacteriol. 2000; 182: 6921-6926Google Scholar, 24Zhang H.B. Wang L.H. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4638-4643Google Scholar). Genetically modified E. carotovora and P. aeruginosa expressing AHL-lactonase or AHL-acylase showed decreased production of virulence factors and attenuated virulence (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar, 18Dong Y.H. Wang L.H. Xu J.L. Zhang H.B. Zhang X.F. Zhang L.H. Nature. 2001; 411: 813-817Google Scholar, 21Reimmann C. Ginet N. Michel L. Keel C. Michaux P. Krishnapillai V. Zala M. Heurlier K. Triandafillu K. Harms H. Defago G. Haas D. Microbiology. 2002; 148: 923-932Google Scholar, 22Lin Y.H. Xu J.L. Hu J. Wang L.H. Ong S.L. Leadbetter J.R. Zhang L.H. Mol. Microbiol. 2003; 47: 849-860Google Scholar). Plants expressing AHL-lactonase quenched pathogen QS signaling and showed significantly enhanced resistance to E. carotovora infection (18Dong Y.H. Wang L.H. Xu J.L. Zhang H.B. Zhang X.F. Zhang L.H. Nature. 2001; 411: 813-817Google Scholar). These findings highlight the promising potential for establishing a generic "quorum-quenching" approach to control bacterial infections, that is, to paralyze the quorum-sensing of bacterial pathogens through inactivation of QS systems (18Dong Y.H. Wang L.H. Xu J.L. Zhang H.B. Zhang X.F. Zhang L.H. Nature. 2001; 411: 813-817Google Scholar, 25Zhang L.H. Trends Plant Sci. 2003; 8: 238-244Google Scholar). AHL-lactonase appears to be a potent enzyme. It works well at physiologically 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 (18Dong Y.H. Wang L.H. Xu J.L. Zhang H.B. Zhang X.F. Zhang L.H. Nature. 2001; 411: 813-817Google Scholar). The enzyme contains a conserved short sequence 104HXHXDH109, which is identical to the Zn2+-binding motif of several metallohydrolases (26Melino S Capo C. Dragani B. Aceto A. Petruzzelli R. Trends Biochem. Sci. 1998; 23: 381-382Google Scholar, 27Carfi A. Pares S Duee E. Galleni M. Duez C. Frere J.M. Dideberg O. EMBO J. 1995; 14: 4914-4921Google Scholar, 28Crowder M.W. Maiti M.K. Banovic L. Makaroff C.A. FEBS Lett. 1997; 418: 351-354Google Scholar). Within the sequence, three amino acid residues, His-106, Asp-108, and His-109, plus His-169, which is also conserved in the metallohydrolases (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar), have proven to be essential for the AHL-lactonase activity (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar, 19Dong Y.H. Gusti A.R. Zhang Q. Xu J.L. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 1754-1759Google Scholar). It is not clear whether Zn2+ or other ions are required for the catalytic function of AHL-lactonase. Little is known about its 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 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 conformational analysis using circular dichroism spectrometry in an attempt to reveal the structural features governing substrate-enzyme interaction and catalytic efficiencies. Synthesis of AHLs and Derivatives—AHLs were synthesized as described (6Zhang L.H. Murphy P.J. Kerr A. Tate M.E. Nature. 1993; 362: 446-448Google Scholar), except that 3-hydroxylbutanoyl l-homoserine lactone was purchased from Quorum Sciences Inc. The acyl homoserines (Acyl-HSs) were prepared by incubating the corresponding acyl homoserine lactones in 1:1 ratio of 1 m NaOH/dimethyl sulfoxide (v/v) for 12 h at room temperature. The solution was neutralized to pH 6.5 with 1 m NaH2PO4 and then dried under vacuum. These synthetic AHLs and Acyl-HSs were purified using silica gel column chromatography and C18 reservephase HPLC and confirmed structurally by 1H NMR spectroscopy and electrospray ionization mass spectrometry. Other reagents were purchased from Sigma-Aldrich unless otherwise stated. Purification of AHL-lactonase and Its Variants—The aiiA gene encoding AHL-lactonase and its variants H106S, D108S, H109S, and H169S contained in the pGEM-7Zf(+) vector were amplified, respectively, by PCR using forward primer 5′-ATCGGATCCATGACAGTAAAGAAGCTTTATTTCG-3′ and reverse primer 5′-GTCGAATTCCTCAACAAGATACTCCTAATGATGT-3′ (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar). The PCR products were digested by BamHI and EcoRI and fused in-frame to the glutathione S-transferase (GST) gene under the control of the isopropyl β-d-thiogalactopyranoside (IPTG)-inducible tac promoter in GST fusion vector pGEX-2T (Amersham Biosciences). The constructs were verified by DNA sequencing. Escherichia coli containing different constructs was cultured at 30 °C in 5-liter LB medium containing 100 μg·ml-1 ampicillin. The GST-AHL-lactonase fusion protein and its variants were expressed by the 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 in 1× PBS buffer (pH 7.4), were disrupted twice with a chilled French pressure cell at 2000 p.s.i. 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 (Amersham Biosciences). 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 room temperature. After digestion, the eluates containing AHL-lactonase were combined, and the purity was analyzed by 10% 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, ϵAHL-lactonase)= 18970 m-1·cm-1. AHL Hydrolysis and Product Analysis—The purified AHL-lactonase (0.4 μm) was mixed with AHL (1 mm) in 1 ml of 0.1 m phosphate buffer (pH 8), and the mixture was incubated at 28 °C for 10 min with gentle shaking. After incubation, the mixture was immediately analyzed by HPLC and electrospray ionization mass spectrometry, using the same conditions described previously (18Dong Y.H. Wang L.H. Xu J.L. Zhang H.B. Zhang X.F. Zhang L.H. Nature. 2001; 411: 813-817Google Scholar). 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. Ali-quots 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 (18Dong Y.H. Wang L.H. Xu J.L. Zhang H.B. Zhang X.F. Zhang L.H. Nature. 2001; 411: 813-817Google Scholar). AHL-lactonase activity was defined as the hydrolyzed micromoles of AHL per minute per microgram 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 to pH 9. 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, and 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 examined by incubating AHL-lactonase solution (3 μm) 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. Metal Ion Determination—The metal ion composition of AHL-lactonase were determined on a PerkinElmer Life Sciences 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. Circular Dichroism 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 path length quartz cells. The spectra were derived from an average of five 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 the absence of enzyme. The fraction of secondary structure was estimated using the method described previously (29Yang J.T. Wu C.-S.C. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Google Scholar). Enzyme Kinetics and Specificity of AHL-lactonase—To determine enzyme kinetics, AHL-lactonase was added at a final concentration of 1 μm to AHL solution (0.3–20 mm)in0.1 m phosphate buffer (pH 7.4) with a final volume of 96 μl. The reactions were incubated at 22 °C, stopped by adding 24 μl 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 was determined by the same procedure, except that the substrate and enzyme concentrations were fixed at 3 mm and 0.5 μm, 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 μm enzyme and 10–200 μm 3-oxo-C8-HSL were transferred to Centricon-10 tubes (Amicon, Millipore) and centrifuged at 5000 × g until the volume of concentrate was ∼60–80 μl. The concentrations of 3-oxo-C8-HSL in the final concentrate and the filtrate were quantified separately by bioassay analysis. 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 and Ser) at the N terminus than does the native AiiA, was separated from GST by thrombin digestion. The recombinant enzyme was purified 86-fold with a yield of ∼7.3% of the total proteins. The SDS-PAGE analysis indicates that the purity of the obtained recombinant AHL-lactonase (7 μg loaded) should be >98.5%, because staining with Coomassie Brilliant Blue R-250, which can detect as little as 0.1 μg of protein, did not reveal other protein bands (Fig. 2). The SDS-PAGE analysis showed the size of the purified AHL-lactonase enzyme is ∼28 kDa, which is consistent with the predicted molecular mass of 28,036 Da (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar). 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 to 8, reached the maximum at pH 8, then declined slightly at pH 9 (Fig. 3A). The potential interference 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 or below. AHL-lactonase exhibited excellent thermal stability at temperatures below 37 °C, and the purified enzyme, kept at 4 and 21 °C for 10 days, still maintained >99% activity. But the enzyme is less stable at higher temperatures; its activity was decreased sharply after incubation for 2 h at >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 °C, the enzyme activity displayed typical temperature dependence as shown in the Arrhenius plot in Fig. 3B, whereas at 45 °C enzyme inactivation was noticed. In the range between 6–37 °C, the activation energy Ea was calculated to be 52.4 kJ·mol-1 from the slope (Ea/R) of the graph. The enthalpy ΔH* and entropy ΔS* of activation were calculated to be 49.9 kJ·mol-1 and -53.7 J·mol-1·K-1, respectively. The free energy ΔG* of activation at 25 °C was calculated to be 65.9 kJ·mol-1. These thermodynamic parameters were calculated by the equations ΔG* = -RTln(kcath/kBT), ΔH* = Ea - RT, and ΔS* = (ΔH* - ΔG*)/T, where kB, h, and R are Boltzmann, Plank, and universal gas constants, respectively. Several metal ions, including Mg2+,Ca2+,Mn2+,Co2+,Ni2+, Zn2+, and Cd2+, showed no effect on enzyme activity at 0.2 and 2mm, 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 of sulfhydryl groups of the enzyme with Cu2+ and Ag+ (30Lehninger A.L. Nelson D.L. Cox M.M. Principles of Biochemistry, 2nd Ed. Worth Publishers, New York,1993: 198-239Google Scholar). 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 its isoelectric point at 4.7 (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar). 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 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 to 9 and slightly changed at pH 6, but significantly changed at pH 5.5 and completely lost at pH 5. 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 (17Dong Y.H. Xu J.L. Li X.Z. Zhang L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3526-3531Google Scholar). To determine whether it is a metalloenzyme, we measured Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Cu2+, Zn2+, and Pb2+ metal ion contents in AHL-lactonase by ICP-MS. Surprisingly, no metal ion was detectable except for a trace mount of Zn2+. The zinc content was ∼0.08 mol per mol of protein, inconsistent with the notion that it is a metallohydrolase. Moreover, the zinc-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 the 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 an excellent ability to accommodate these structural differences regardless of which AHL was used as a substrate. More than 80% of 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 new fraction showed the identical HPLC retention time and molecular mass as the corresponding 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 (31Byers J.T. Lucas C. Salmond G.P.C. Welch M. J Bacteriol. 2002; 184: 1163-1171Google Scholar, 32Yates E.A. Philipp B. Buckley C. Atkinson S. Chhabra S.R. Sockett R.E. Goldner M. Dessaux Y. Camara M. Smith H. Williams P. Infect. Immun. 2002; 70: 5635-5646Google Scholar), was negligible in this time scale. The AHL lactonolysis by AHL-lactonase led to a sharp decrease (>1500-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 (33Piper K.R. Beck von Bodman S. Farrand S.K. Nature. 1993; 362: 448-450Google Scholar). 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 1 week.Table IBiological activity of AHL hydrolysis products Samples were diluted, and the minimum concentration of each AHL hydrolysis product (Acyl-HS) required to induce formation of a visible blue colony on the bioassay plate was presented. The data were means of at least three repeats.AHL hydrolysis productsMinimum concentration for activityaND indicates that no activity was detected in the concentration range from 10-10,000 μmDecreased activity (times) compared with the corresponding AHLμm3-Oxo-C4-HSND3-Oxo-C6-HS50016673-Oxo-C8-HS5025003-Oxo-C10-HS30015003-Oxo-C12-HS10001667C4-HSNDC6-HSNDC8-HS10002000C10-HS200020003-HO-C4-HSb3-Hydroxylbutanoyl l-homoserineNDa ND indicates that no activity was detected in the concentration range from 10-10,000 μmb 3-Hydroxylbutanoyl l-homoserine Open table in a new tab 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 a 1:6000 molar ratio and incubated for 10 min. Fig. 5 shows that the AHL-lactonase exhibited high relative activities toward all of the tested AHLs. Differences in acyl chain length and substitution did not significantly affect the enzyme activity, indicating that AHL-lactonase has a broad catalytic spectrum toward AHLs. Within a narrow margin, AHL-lactonase worked better against AHLs without 3-oxo substitution than did 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-lactonase showed little activity toward γ-decanolactone, which has a 6-carbon alkane side chain. Several lactonases were also known to digest p-nitrophenyl acetate (34Hucho F. Wallenfels K. Biochim. Biophys. Acta. 1972; 276: 176-179Google Scholar), which is a non-cyclic ester substrate. However, AHL-lactonase did not hydrolyze any of the tested non-cyclic esters, including ethyl acetate, phenyl acetate, p-nitrophenyl acetate, and α-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 showed comparable catalytic activity against a range of structurally different AHLs with kcat and Km values ranging 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 displayed higher kcat and kcat/Km values against the AHLs with fully reduced acyl chains than their corresponding derivatives containing 3-oxo substitution (Table II).Tabled 1AHLskcataThe data are means from triplicate experimentsKmaThe data are means from triplicate experimentskcat/KmaThe data are means from triplicate experimentss-1mmmm-1·s-13-Oxo-C4-HSL28.634.077.033-Oxo-C6-HSL22.682.957.693-Oxo-C8-HSL22.172.289.723-Oxo-C10-HSL20.221.4314.13-Oxo-C12-HSLNDbND indicates not determined due to poor solubility of the substrate in phosphate bufferNDNDC4-HSL37.635.117.36C6-HSL35.673.839.31C8-HSL27.532.6110.5C10-HSLNDbND indicates not de
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