Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures

Enzymes capable of hydrolyzing N-acyl- l-homoserine lactones (AHLs) used in some bacterial quorum-sensing pathways are of considerable interest for their ability to block undesirable phenotypes. Most known AHL hydrolases that catalyze ring opening (AHL lactonases) are members of the metallo-beta-lactamase enzyme superfamily and rely on a dinuclear zinc site for catalysis and stability. Here we report the three-dimensional structures of three product complexes formed with the AHL lactonase from Bacillus thuringiensis. Structures of the lactonase bound with two different concentrations of the ring-opened product of N-hexanoyl- l-homoserine lactone are determined at 0.95 and 1.4 A resolution and exhibit different product configurations. A structure of the ring-opened product of the non-natural N-hexanoyl- l-homocysteine thiolactone at 1.3 A resolution is also determined. On the basis of these product-bound structures, a substrate-binding model is presented that differs from previous proposals. Additionally, the proximity of the product to active-site residues and observed changes in protein conformation and metal coordination provide insight into the catalytic mechanism of this quorum-quenching metalloenzyme.     

The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein

Lactonases from Bacillus species hydrolyze the N-acylhomoserine lactone (AHL) signaling molecules used in quorum-sensing pathways of many Gram-negative bacteria, including Pseudomonas aeruginosa and Erwinia carotovora, both significant pathogens. Because of sequence similarity, these AHL lactonases have been assigned to the metallo-beta-lactamase superfamily of proteins, which includes metalloenzymes of diverse activity, mechanism, and metal content. However, a recent study claims that AHL lactonase from Bacillus sp. 240B1 is not a metalloprotein [Wang, L. H., et al. (2004) J. Biol. Chem. 279, 13645]. Here, the gene for an AHL lactonase from Bacillus thuringiensis is cloned, and the protein is expressed, purified, and found to bind 2 equiv of zinc. The metal-bound form of AHL lactonase catalyzes the hydrolysis of N-hexanoyl-(S)-homoserine lactone but not the (R) enantiomer. Removal of both zinc ions results in loss of activity, and reconstitution with zinc restores activity, indicating the importance of metal ions for catalytic activity. Metal content, sequence alignments, and X-ray absorption spectroscopy of the zinc-containing lactonase all support a proposed dinuclear zinc binding site similar to that found in glyoxalase II.     

The role of AiiA, a quorum-quenching enzyme from Bacillus thuringiensis, on the rhizosphere competence

Bacteria sense their population density and coordinate the expression of target genes, including virulence factors in Gram-negative bacteria, by the N-acylhomoserine lactones (AHLs)-dependent quorum-sensing (QS) mechanism. In contrast, several soil bacteria are able to interfere with QS by enzymatic degradation of AHLs, referred to as quorum quenching. A potent AHL-degrading enzyme, AiiA, of Bacillus thuringiensis has been reported to effectively attenuate the virulence of bacteria by quorum quenching. However, little is known about the role of AiiA in B. thuringiensis itself. In the present study, an aiiA-defective mutant was generated to investigate the role of AiiA in rhizosphere competence in the root system of pepper. The aiiA mutant showed no detectable AHL-degrading activity and was less effective for suppression of soft-rot symptom caused by Erwinia carotovora on the potato slice. On the pepper root, the survival rate of the aiiA mutant significantly decreased over time compared with that of wild type. Interestingly, viable cell count analysis revealed that the bacterial number and composition of E. carotovora were not different between treatments of wild type and the aiiA mutant, although root application of the aiiA mutant in pepper failed to protect the plant from root rot. These results provide evidence that AiiA can play an important role in rhizosphere competentce of B. thuringiensis and bacterial quorum quenching to Gram-negative bacteria without changing bacterial number or composition.     

Structure and specificity of a quorum-quenching lactonase (AiiB) from Agrobacterium tumefaciens

N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called "quorum-quenching" enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.     

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k_cat/K_m of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.     

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.     

Purification and characterization of the active fragment from Bacillus thuringiensis delta-toxin

Limited tryptic hydrolysis of a partially purified delta-toxin (Mr = 100,000) from Bacillus thuringiensis, has produced a polypeptide fragment of Mr = 60,000 containing the full biological activity. The fragment was the only polypeptide observed in the polyacrylamide-gel electrophoresis of the delta-toxin after treatment with trypsin and could be purified by DEAE-cellulose chromatography. Amino acid and partial sequence analyses indicate that the 60,000 Mr fragment has been derived from the mid-section of the holotoxin peptide; over 80% of Lys, 65% of Pro and 50% of His residues in the holotoxin have been lost in the active fragment. This section must contain the active site since its specific insecticidal activity is approximately twice that of the holotoxin. The active fragment shows complete cross-reactivity with the antiserum raised against the native toxin, and appeared to possess higher thermal stability than the mother protein. It provides a powerful tool for studies of the structure involved in the insecticidal activity.     

Characterization of the PON1 active site using modeling simulation, in relation to PON1 lactonase activity

Paraoxonase1 (PON1) is a HDL bound enzyme and many of the anti-atherogenic properties of HDL are attributed to PON1. The enzyme precise mechanism of protective action and its endogenous substrate remain elusive. PON1 hydrolyzes organophosphates, arylesters and lactones, whereas the lactones activity is assumed as the physio/pathological one. This study is aimed to predict the location of the PON1 active site within PON1 crystal structure, and the lactone structure suitability as PON1 ligand, by employing modeling techniques. Based on such calculations the ligands-PON1 interactions were characterized, and relating lactones rate of hydrolysis revealed an inverse correlation with the docking energy of the ligands-PON1 complex, and a direct correlation with the lactone side chain length. In conclusion, this study characterized the PON1 possible active site and proposes a tool which may make it possible to envisage the structure of potential endogenous and exogenous lactones such as the PON1 ligand.     

Inhibition of Vibrio cholerae biofilm by AiiA enzyme produced from Bacillus spp.

Vibrio cholerae is the causative agent of water-borne diarrheal disease, cholera. The formation of biofilm favors survival and persistence of V. cholerae in the aquatic environment and also inside the host. AHL lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus spp., blocks quorum sensing in Gram-negative bacteria by hydrolyzing N-acyl-homoserine lactones (AHLs). In the present investigation, AiiA-mediated inhibition of V. cholerae biofilm was studied. Two novel alleles of aiiA-encoding genes from Bacillus spp. were expressed in E. coli, and the results demonstrated that AiiA enzyme is a potent inhibitor of V. cholerae biofilm.     

Substrate binding characteristics of the active site of spermidine dehydrogenase from Serratia marcescens.

The substrate specificity of spermidine dehydrogenase from Serratia marcescens was studied using many kinds of naturally occurring and synthetic polyamines. Diamines inhibited the enzyme competitively and their inhibitor constants tended to decrease with increasing methylene chain length in the diamines. All of the triamines and tetramines examined were active as substrates, and the amines containing a 4-aminobutylimino moiety (NH2(CH2)4NH-) in their structures were more active. N-Alkylputrescine was also oxidized by the enzyme. All of the amines containing a 4-aminobutylimino group were oxidized to form 1-pyrroline stoichiometrically as one of the products. Tetramines containing a 3-aminopropylimino group (NH2(CH2)3NH-) were oxidized to form 1,3-diaminopropane. However, in the case of an amine containing both 4-aminobutylimino and 3-aminopropylimino groups, the imino moiety of the former was preferentially oxidized by the enzyme. On the basis of the substrate specificity, the binding characteristics of the enzyme are discussed and a subsite model for the binding site is proposed.     

A novel strain of Bacillus thuringiensis (NCIMB 40152) active against coleopteran insects

Abstract: A novel Bacillus thuringiensis strain, NCIMB 40152, was isolated from dead Tenebrio molitor L. larvae. The isolate is endowed with specific insecticide activity against the Colorado potato beetle ( Leptinotarsa decemlineata (Say), Coleoptera, Crysomelidae), but has no effect against lepidopteran or dipteran insects.
During the sporulation process it produces diamond-shaped crystals and small inclusions. Rare flat, square crystals are alsò produced. The strain can be differentiated from the other reported B. thuringiensis strains active against Coleoptera on the basis of physiological and biochemical tests, flagellar serotype and toxin electrophoretic patterns.
NCIMB 40152 toxin is easily extractable from spore/crystal preparations in neutral distilled water without detergents or reducing agents.     

Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis

To investigate the roles of the active site residues in the catalysis of Bacillus thuringiensis WB7 chitinase, twelve mutants, F201L, F201Y, G203A, G203D, D205E, D205N, D207E, D207N, W208C, W208R, E209D and E209Q were constructed by site-directed mutagenesis. The results showed that the mutants F201L, G203D, D205N, D207E, D207N, W208C and E209D were devoid of activity, and the loss of the enzymatic activities for F201Y, G203A, D205E, W208R and E209Q were 72, 70, 48, 31 and 29%, respectively. The pH-activity profiles indicated that the optimum pH for the mutants as well as for the wildtype enzyme was 8.0. E209Q exhibited a broader active pH range while D205E, G203A and F201Y resulted in a narrower active pH range. The pH range of activity reduced 1 unit for D205E, and 2 units for G203A and F201Y. The temperature-activity profiles showed that the optimum temperature for other mutants as well as wildtype enzyme was 60°C, but 50°C for G203A, which suggested that G203A resulted in a reduction of thermostability. The study indicated that the six active site residues involving in mutagenesis played an important part in WB7 chitinase. In addition, the catalytic mechanisms of the six active site residues in WB7 chitinase were discussed.     

Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.     

Computational tridimensional protein modeling of Cry1Ab19 toxin from Bacillus thuringiensis BtX-2

We report the computational structural simulation of the Cry1Ab19 toxin molecule from B. thuringiensis BtX-2 based on the structure of Cry1Aa1 deduced by x-ray diffraction. Validation results showed that 93.5% of modeled residues are folded in a favorable orientation with a total energy Z-score of -8.32, and the constructed model has an RMSD of only 1.13. The major differences in the presented model are longer loop lengths and shortened sheet components. The overall result supports the hierarchical three-domain structural hypothesis of Cry toxins and will help in better understanding the structural variation within the Cry toxin family along with facilitating the design of domain-swapping experiments aimed at improving the toxicity of native toxins.     

Gentisate 1,2-dioxygenase from Pseudomonas. Substrate coordination to active site Fe2+ and mechanism of turnover

Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate. The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex. Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand. Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to [17O]water to disappear, suggesting that water is displaced. Hyperfine broadening of the EPR spectrum for the gentisate-bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent. Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron. Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands. The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni. Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate. Furthermore, an electron-donating group that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover. The best alternate substrate of this group is 5-aminosalicylate, which is turned over at approximately 7% of the rate of gentisate by the enzyme from P. testosteroni. Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover. This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase. It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron. Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.     

The potential active site of the lipoprotein-specific (type II) signal peptidase of Bacillus subtilis.

Type II signal peptidases (SPase II) remove signal peptides from lipid-modified preproteins of eubacteria. As the catalytic mechanism employed by type II SPases was not known, the present studies were aimed at the identification of their potential active site residues. Comparison of the deduced amino acid sequences of 19 known type II SPases revealed the presence of five conserved domains. The importance of the 15 best conserved residues in these domains was investigated using the type II SPase of Bacillus subtilis, which, unlike SPase II of Escherichia coli, is not essential for viability. The results showed that only six residues are important for SPase II activity. These are Asp-14, Asn-99, Asp-102, Asn-126, Ala-128, and Asp-129. Only Asp-14 was required for stability of SPase II, indicating that the other five residues are required for catalysis, the active site geometry, or the specific recognition of lipid-modified preproteins. As Asp-102 and Asp-129 are the only residues invoked in the known catalytic mechanisms of proteases, we hypothesize that these two residues are directly involved in SPase II-mediated catalysis. This implies that type II SPases belong to a novel family of aspartic proteases.