Altering substrate chain length specificity of an acylhomoserine lactone synthase in bacterial communication

Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the "right" acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expI(SCC1) identified the changes M127T and F69L as sufficient to effectively alter ExpI(SCC1) (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language.     

A single mutation in P450BM-3 enhances acyl homoserine lactone: acyl homoserine substrate binding selectivity nearly 250-fold

Quorum sensing is the process by which bacteria alter gene regulation in response to their population density. The enzymatic inactivation of quorum signals has shown promise for use in genetically modified organisms resistant to pathogens. We recently characterized the ability of a cytochrome P450, P450BM-3, to oxidize the quorum sensing signals known as acyl homoserine lactones. The oxidation of the acyl homoserine lactones reduced their activity as quorum signals. The enzyme also oxidized the inactive lactonolysis products, acyl homoserines. The enzyme showed similar binding affinity for the acyl homoserine lactones and acyl homoserines. The latter reaction may lead to problems when lactonases and the P450-dependent system are used in tandem, as oxidation of the acyl homoserines produced by lactonolysis in vivo may compete with acyl homoserine lactone oxidation by the cytochrome P450. We report here that a single mutation (R47S) in P450BM-3 is capable of increasing the acyl homoserine lactone: acyl homoserine substrate binding selectivity of the enzyme nearly 250-fold, reducing the potential for competition by acyl homoserines and significantly enhancing the potential for use of P450BM-3 as part of a pathogen resistance system in genetically modified crops.     

Proline-valine pseudo peptide enol lactones. Effective and selective inhibitors of chymotrypsin and human leukocyte elastase

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.     

Antiprotozoal activity of 8-acyl and 8-alkyl incomptine A analogs

The activities of 11 C-8-O-acyl and alkyl analogs of the antiprotozoal sesquiterpene lactone, incomptine A (1) against Entamoeba histolytica and Giardia lamblia, were determined. Here, the effects of different lengths and amounts of branching of the acyl and alkyl groups on the antiprotozoal activity of the synthesized incomptine A-analogs are reported.     

Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase)

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 (104)HX- HXDH(109) 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 (106)HXDH(109) approximately H(169) of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity.     

New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing

A series of 22 novel synthetic N-acyl-homoserine lactone analogues has been evaluated for both their inducing activity and their ability to competitively inhibit the action of 3-oxo-hexanoyl-L-homoserine lactone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri. In the newly synthesized analogues, the extremity of the acyl chain was modified by introducing ramified alkyl, cycloalkyl or aryl substituents at the C-4 position. Most of the analogues bearing either acyclic or cyclic alkyl substituents showed inducing activity. In contrast, the phenyl substituted analogues displayed significant antagonist activity. We hypothesized that the antagonist activity of the phenyl compounds may result from the interaction between the aryl group and aromatic amino acids of the LuxR receptor, preventing it from adopting the active dimeric form.     

Reaction of acylated homoserine lactone bacterial signaling molecules with oxidized halogen antimicrobials.

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.     

Isolation and characterization of N-acylhomoserine lactonase from the thermophilic bacterium, Geobacillus caldoxylosilyticus YS-8

Geobacillus caldoxylosilyticus YS-8, which was isolated from volcanic soil in Indonesia, was found to degrade various N-acylhomoserine lactones (AHLs) with different lengths and acyl side-chain substitutions over a wide temperature range of 30-70 °C. The purified AHL-degrading enzyme showed a single band of 32 kDa, and its N-terminal amino acid sequence was determined to be ANVIKARPKLYVMDN, tentatively suggesting that the AHL-degrading enzyme was AHL lactonase. The AHL-degrading activity of the purified enzyme was maximized at pH 7.5 and 50 °C, and it retained about 50% of its activity even after a heat treatment at 60 °C for 3 h, exhibiting properties consistent with a thermostable enzyme. The mass spectrometric analysis demonstrated that the AHL-degrading enzyme catalyzed lactone ring opening of N-3-oxohexanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone by hydrolyzing the lactones and working as an AHL lactonase.     

Kinetics and mechanism of human leukocyte elastase inactivation by ynenol lactones

Human leukocyte elastase (HLE), a serine protease involved in inflammation and tissue degradation, can be irreversibly inactivated in a time- and concentration-dependent manner by ynenol lactones. Ynenol lactones that are alpha-unsubstituted do not inactivate but are alternate substrate inhibitors that are hydrolyzed by the enzyme. Ynenol lactones that are both substituted alpha to to the lactone carbonyl and unsubstituted at the acetylene terminus are rapid inactivators of HLE and inactivate pancreatic elastase and trypsin more slowly. 3-Benzyl-5(E)-(prop-2-ynylidene)tetrahydro-2-furanone inactivates HLE with biphasic kinetics and an apparent second-order rate of up to 22,000 M-1 s-1 (pH 7.8, 25 degrees C). The rate of inactivation is pH-dependent and is slowed by a competitive inhibitor. The partition ratio is 1.6 +/- 0.1. Rapid removal of ynenol lactone during the course of inactivation yields a mixture of acyl and inactivated enzyme species, which then shows a partial recovery of activity that is time- and pH-dependent. Inactivation is not reversible with hydroxylamine. The enzyme is not inactivated if the untethered allenone is added exogenously. All of these results are consistent with a mechanism involving enzyme acylation at serine-195 by the ynenol lactone, isomerization of the acyl enzyme to give a tethered allenone, and capture of a nucleophile (probably histidine-57) to inactivate the enzyme. Substitution at the acetylene terminus of ynenol lactones severely reduces their ability to inactivate HLE, because allenone formation is slowed and/or nucleophile capture is hindered. Chemical competence of each of these steps has been demonstrated [Spencer, R.W., Tam, T.F., Thomas, E.M., Robinson, V.J.,& Krantz, A. (1986) J. Am. Chem. Soc. 108, 5589-5597].     

Novel roles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> to control plant diseases

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.     

Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.     

Identification of novel long chain N-acylhomoserine lactones of chain length C20 from the marine phototrophic bacterium Rhodovulum sulfidophilum

Gram-negative bacterial quorum sensing is mainly regulated by an extracellularly produced N-acylhomoserine lactone (AHL). AHL consists of a lactone ring and an acyl chain, which generally varies from C₄ to C₁₈ in length and affords species-specific variety. In this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometry system and detected two kinds of long chain AHLs with chain length C₂₀ from the reverse-phase thin layer chromatography-fractionated cultured supernatant of the marine photosynthetic bacterium Rhodovulum sulfidophilum. By fragmentation search analysis to detect compounds with a homoserine lactone ring moiety for data dependent acquisition, a minor AHL, presumed to be 3-OH-C₁₈-homoserine lactone (HSL), was also found. Among the detected C₂₀-HSLs, 3-OH-C₂₀-HSL was structurally identified and 3-OH-C_20:1-HSL was strongly suggested. To our knowledge, this is the first report to show a novel AHL with the longest C₂₀ acyl side chain found to date.

Abbreviations: AGC: automatic gain control; AHL: N-acylhomoserine lactone; CD: cyclodextrin; CID: collision induced dissociation; DDA: data dependent acquisition; EPI: enhanced product ion; FISh: fragment ion search; HCD: high energy collisional dissociation; HSL: homoserine lactone; IT: injection time; LC: liquid chromatography; MS: mass spectrometry; PRM: parallel reaction monitoring; RP: reverse phase; SRM: selected reaction monitoring; TLC: thin layer chromatography; UHPLC: ultra high performance liquid chromatography     

In Vivo Evidence that S-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases

Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the fad regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular S-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine.     

Quorum quenching enzyme activity is widely conserved in the sera of mammalian species

Acyl-homoserine lactone (AHL) quorum sensing signals play a key role in synchronizing virulence gene expression in Pseudomonas aeruginosa, which could cause fatal bloodstream infections. We showed that AHL inactivation activity, albeit with variable efficiency, was conserved in the serum samples of all the 6 tested mammalian animals. High-performance liquid chromatography and mass spectrometry analyses revealed that mammalian sera had a lactonase-like enzyme(s), which hydrolyzed the lactone ring of AHL to produce acyl homoserine, with enzyme properties reminiscent of paraoxonases (PONs). We further showed that the animal cell lines expressing three mouse PON genes, respectively, displayed strong AHL degradation activities.     

Analysis of random and site-directed mutations in rhlI, a Pseudomonas aeruginosa gene encoding an acylhomoserine lactone synthase

The opportunistic human pathogen Pseudomonas aeruginosa possesses two cell density-dependent genetic regulatory systems that control expression of a number of secreted virulence factors. These two systems, the lasI–lasR and rhlI–rhlR gene pairs, are members of the luxI–luxR family of quorum-sensing signal generators and signal receptors. The rhlI gene in P. aeruginosa encodes a 201-amino-acid protein that catalyses the synthesis of an autoinducer, butyrylhomoserine lactone. Through a programme of random and site-specific mutagenesis of rhlI we have gained a better understanding of how its protein product functions. Eight residues critical to butyrylhomoserine lactone synthesis by RhlI were identified by random mutagenesis, and all mapped to a conserved region that spans residues 24–104. Seven of the eight residues were charged amino acids and the other was a glycine. By using site-specific mutagenesis we showed that an active-site cysteine or serine was not required for butyrylhomoserine lactone synthesis, and that two conserved aromatic amino acids in the postulated active site region could be altered without complete loss of RhlI activity. Furthermore, two residues towards the C-terminus that align with critical residues in LuxI can be altered in RhlI without loss of activity. These studies suggest that as opposed to the current models for acyl substrate binding to quorum-sensing signal generators, charged amino acid residues participate directly in the catalysis of butyrylhomoserine lactone synthesis rather than cysteines, serines or hydrophobic amino acids.     

Acyl and alk-1-enyl group compositions of the alk-1'-enyl acyl and the diacyl glycerophosphoryl ethanolamines of mouse and ox brain

The ethanolamine phosphoglycerides were prepared from lipid extracts of ox and mouse brains by preparative thin-layer chromatography. The cyclic acetal derivatives of the alk-1-enyl groups were made by treating the ethanolamine phosphoglycerides with 1,3-propanediol. The resulting monoacyl glycerophosphoryl ethanolamines were separated from the unchanged ethanolamine phosphoglycerides by preparative thin-layer chromatography. Methyl ester derivatives of the acyl groups from both of these fractions were prepared by alkaline methanolysis. The cyclic acetal and methyl ester derivatives were analyzed by gas-liquid chromatography. Substantial differences were found in the composition of the side chains when the combined alk-1-enyl and acyl side chains of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were compared with the side chains of the diacyl glycerophosphoryl ethanolamines. The side chains from the 1-position of these two ethanolamine phosphoglycerides are different in chain length and unsaturation as well as in chemical bonding. The acyl groups from the 2-position of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were predominantly unsaturated. Therefore, acyl group compositions of the total ethanolamine phosphoglyceride from brain are of limited value and individual types should be analyzed.     

Postharvest temperature influences volatile lactone production via regulation of acyl-CoA oxidases in peach fruit

The biosynthesis of volatile compounds in plants is affected by environmental conditions. Lactones are considered to be peach‐like aroma volatiles; however, no enzymes or genes associated with their biosynthesis have been characterized. White‐fleshed (cv. Hujingmilu) and yellow‐fleshed (cv. Jinxiu) melting peach (Prunus persica L. Batsch) fruit were used as materials in two successive seasons and responses measured to four different temperature treatments. Five major lactones accumulated during postharvest peach fruit ripening at 20 °C. Peach fruit at 5 °C, which induces chilling injury (CI), had the lowest lactone content during subsequent shelf life after removal, while 0 °C and a low‐temperature conditioning (LTC) treatment alleviated development of CI and maintained significantly higher lactone contents. Expression of PpACX1 and activity of acyl‐CoA oxidase (ACX) with C16‐CoA tended to increase during postharvest ripening both at 20 °C and during shelf life after removal from cold storage when no CI was developed. There was a positive correlation between ACX and lactones in peach fruit postharvest. Changes in lactone production in response to temperatures are suggested to be a consequence of altered expression of PpACX1 and long‐chain ACX activity.     

Sinapoyltransferases in the light of molecular evolution

Acylation is a prevalent chemical modification that to a significant extent accounts for the tremendous diversity of plant metabolites. To catalyze acyl transfer reactions, higher plants have evolved acyltransferases that accept β-acetal esters, typically 1-O-glucose esters, as an alternative to the ubiquitously occurring CoA-thioester-dependent enzymes. Shared homology indicates that the β-acetal ester-dependent acyltransferases are derived from a common hydrolytic ancestor of the Serine CarboxyPeptidase (SCP) type, giving rise to the name Serine CarboxyPeptidase-Like (SCPL) acyltransferases. We have analyzed structure–function relationships, reaction mechanism and sequence evolution of Arabidopsis 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (AtSMT) and related enzymes to investigate molecular changes required to impart acyltransferase activity to hydrolytic enzymes. AtSMT has maintained the catalytic triad of the hydrolytic ancestor as well as part of the H-bond network for substrate recognition to bind the acyl acceptor l-malate. A Glu/Asp substitution at the amino acid position preceding the catalytic Ser supports binding of the acyl donor 1-O-sinapoyl-β-glucose and was found highly conserved among SCPL acyltransferases. The AtSMT-catalyzed acyl transfer reaction follows a random sequential bi-bi mechanism that requires both substrates 1-O-sinapoyl-β-glucose and l-malate bound in an enzyme donor–acceptor complex to initiate acyl transfer. Together with the strong fixation of the acyl acceptor l-malate, the acquisition of this reaction mechanism favours transacylation over hydrolysis in AtSMT catalysis. The model structure and enzymatic side activities reveal that the AtSMT-mediated acyl transfer proceeds via a short-lived acyl enzyme complex. With regard to evolution, the SCPL acyltransferase clade most likely represents a recent development. The encoding genes are organized in a tandem-arranged cluster with partly overlapping functions. With other enzymes encoded by the respective gene cluster on Arabidopsis chromosome 2, AtSMT shares the enzymatic side activity to disproportionate 1-O-sinapoyl-β-glucoses to produce 1,2-di-O-sinapoyl-β-glucose. In the absence of the acyl acceptor l-malate, a residual esterase activity became obvious as a remnant of the hydrolytic ancestor. With regard to the evolution of Arabidopsis SCPL acyltransferases, our results suggest early neofunctionalization of the hydrolytic ancestor toward acyltransferase activity and acyl donor specificity for 1-O-sinapoyl-β-glucose followed by subfunctionalization to recognize different acyl acceptors.     

Medium sized lactones with hypolipidaemic and antioxidant activity: synthesis and biological evaluation of promising dual-action anti-atherosclerosis drugs

Macrocyclic lactones 1a-b have been synthesized and their potential therapeutic value evaluated. The key structural feature of these active 'chimera' compounds is the 12-membered lactone ring that brings together the well-known polysubstituted hydroquinone moiety of antioxidants and the alpha,alpha-dimethyl substituted acyl residue of gemfibrozil. Lactones 1a-b showed better activity than probucol, a classical phenolic antioxidant, in preventing the Cu++-induced oxidative modification of human LDL. The hypolipidaemic activity of the new lactones, evaluated as the inhibition of lipids biosynthesis in Hep-G2 cells, was comparable to that of gemfibrozil. These features, added to the lack of cytotoxicity, make this new class of medium sized lactones promising dual-action drugs useful as anti-atherosclerosis agents.