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.     

Alternate substrate inhibitors of an alpha-chymotrypsin: enantioselective interaction of aryl-substituted enol lactones

Four enol lactones, bearing phenyl or 1-naphthyl substituents on the alpha or beta positions [3-phenyl-6-methylenetetrahydro-2-pyranone (alpha Ph6H, IIc), 3-(1-naphthyl)-6-methylenetetrahydro-2-pyranone (alpha Np6H, IId), 4-phenyl-6-methylenetetrahydro-2-pyranone (beta Ph6H, IIIc), and 4-(1-naphthyl)-6-methylenetetrahydro-2-pyranone (beta Np6H, IIId)], available as pure R and S enantiomers, have been studied as alternate substrate inhibitors of chymotrypsin. Kinetic constants for substrate binding (Ks) and acylation (ka) were determined by a competitive substrate assay, using succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide; the deacylation rate constant (kd) was determined by the proflavin displacement assay. All lactones undergo rapid acylation (ka varies from 17 to 170 min-1) that shows little enantioselectivity; there is, however, pronounced enantioselectivity in substrate binding for three of the lactones [Ks(R/S) = 40-110]. In each case it is the enantiomer with the S configuration that has the higher affinity. In all cases, deacylation rates are slow, and in two cases, acyl enzymes with half-lives of 4.0 and 12.5 h at pH 7.2, 25 degrees C, are obtained (for beta Ph6H and alpha Np6H, respectively). In these cases, high deacylation enantioselectivity is observed [kd(S/R) = 60-70], and the lactone more weakly bound as a substrate (R enantiomer) gives the more stable acyl enzyme. Two hypotheses, involving hindrance of the attack of water or an exchange of the ester and ketone carbonyl groups in the acyl enzyme, are advanced as possible explanations for the high stability of these acyl enzymes.     

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.     

Halo enol lactones: studies on the mechanism of inactivation of alpha-chymotrypsin

In a previous investigation [Daniels, S. B., Cooney, E., Sofia, M. J., Chakravarty, P. K., & Katzenellenbogen, J. A. (1983) J. Biol. Chem. 258, 15046-15053], we demonstrated that alpha-aryl-substituted five- and six-membered ring halo enol lactones were effective inhibitors of chymotrypsin, and we proposed that they reacted by an enzyme-activated mechanism: acyl transfer to the active site serine generates a halomethyl ketone that remains tethered in the catalytic site until it alkylates an accessible nucleophilic residue. In this study, we have investigated in greater detail the process of chymotrypsin inactivation by an alpha-naphthyl-substituted five- and six-membered bromo enol lactone. Inactivation by both compounds appears to be active site directed, since the time-dependent inactivation is retarded by competing substrate. The possible involvement of a paracatalytic mechanism for inactivation (generation of a free, rather than active site bound, inactivating species) was investigated by comparing the inactivation efficiencies of the lactones with that of the bromomethyl keto acid hydrolysis products. The bromomethyl ketone derived from the five-membered lactone is ineffective, whereas that derived from the six-membered lactone is highly efficient. However, the possible involvement of the free keto acid in chymotrypsin inactivation by the six-membered lactone is ruled out by experiments involving selective scavenging. The long-term inactivation of chymotrypsin requires the presence of the bromine substituent and appears to involve an alkylation rather than an acylation reaction (hydrazine resistant). Furthermore, a 1:1 lactone:enzyme stoichiometry is demonstrated with the 14C-labeled six-membered lactone. These results are consistent with the mechanism-based inactivation process previously presented.     

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].     

Distribution of enantiomers of volatile organic compounds in selected fruit distillates

The enantiomer ratios of chiral volatile organic compounds in fruit distillates were determined by multidimensional gas chromatography using solid‐phase microextraction (SPME) as a sample treatment procedure. Linalool and its oxides, limonene, α‐terpineol, and nerolidol, were present at the highest concentration levels, while significantly lower amounts of β‐citronellol and lactones were found in the studied samples. However, almost all terpenoids mainly occur as a racemic or near‐racemic mixture; enantiomer distribution of some chiral organic compounds in fruit distillates correlated to a botanical origin. In particular, a significant enantiomeric excess of (R)‐linalool and (S)‐α‐terpineol was found only for pear brandy, and likewise the dominance (R)‐limonene and the second eluted enantiomer of nerolidol for Sorbus domestica and strawberry, respectively. The distribution of γ‐lactones stereoisomers was more nonspecific, with a general excess of the R‐enantiomer.     

Comparison of cardiolipins from Drosophila strains with mutations in putative remodeling enzymes

Cardiolipin is a dimeric phospholipid with a characteristic acyl composition that is generated by fatty acid remodeling after de novo synthesis. Several enzymes have been proposed to participate in acyl remodeling of cardiolipin. In order to compare the effect of these enzymes, we determined the pattern of cardiolipin molecular species in Drosophila strains with specific enzyme deletions, using MALDI-TOF mass spectrometry with internal standards. We established the linear range of the method for cardiolipin quantification, determined the relative signal intensities of several cardiolipin standards, and demonstrated satisfying signal-to-noise ratios in cardiolipin spectra from a single fly. Our data demonstrate changes in the cardiolipin composition during the Drosophila life cycle. Comparison of cardiolipin spectra, using vector algebra, showed that inactivation of tafazzin had a large effect on the molecular composition of cardiolipin, inactivation of calcium-independent phospholipase A(2) had a small effect, whereas inactivation of acyl-CoA:lysocardiolipin-acyltransferase and of the trifunctional enzyme did not affect the cardiolipin composition.     

Interaction of clavulanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The reactions of beta-lactamases of Actinomadura R39 and Streptomyces albus G with clavulanate proceed along branched pathways. Both enzymes perform the hydrolysis of this beta-lactam with rather high efficiencies (kcat. = 18s-1 and 52s-1 respectively). If large clavulanate/enzyme ratios are used, complete inactivation of the enzymes is observed. At lower ratios, inactivation is only partial. Irreversible inactivation occurs after 400 and 20000 turnovers for the A. R39 and S. albus G enzymes respectively. With the A. R39 beta-lactamase, a transiently inhibited complex is also formed that remains undetectable with the S. albus G beta-lactamase. Kinetic models are presented and studied for the interaction between clavulanate and both enzymes. A tentative general reaction scheme is also discussed.     

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.     

Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

A monoterpene epsilon-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30 degrees C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.     

Lactones of methyl 3-O

Lactones of methyl 3-O-[(R)- and (S)-1-carboxyethyl]-alpha-D-gluco-, galacto- and manno-pyranoside were prepared by treatment of the sugar derivatives in acetic acid. The lactones were formed between the 1-carboxyethyl substituent and 2-OH or 4-OH in different proportions depending on the stereochemistry of the parent compounds. Relative formation rates in acetic acid-d4 and hydrolysis rates in buffered D2O solutions at pD 2.4, 4.6 and 7.4 were estimated. Hydrolysis of the formed lactones is relatively slow in D2O at pD 4.6, which permitted characterization of the lactones by 1H and 13C NMR spectroscopy in buffered D2O solutions. Hydrolysis of the lactones in 1 M aqueous NaOH at 80 degrees C gave no detectable isomerization of the alpha-carbon. The set of lactones formed from the 1-carboxyethyl substituted methyl glycosides used in this study showed large similarities in the NMR shifts (delta delta values). Deviations from the observed shift pattern were found for two lactones. Our findings strongly suggest that those two lactones differ from the rest by adopting a boat-like conformation, whereas the others adopt pseudo-chair conformations.     

The beta-lactamase of Streptomyces cacaoi: interaction with cefoxitin and beta-iodopenicillanate

Cefoxitin was a very poor substrate for the beta-lactamase of Streptomyces cacaoi (kcat = 2.7 x 10(-4) s-1). In the presence of nitrocefin, a good substrate, cefoxitin behaved as a transient inactivator by immobilizing a large proportion of the enzyme as the acyl enzyme intermediate. The enzyme was also inactivated by beta-iodopenicillanate. In this case, the acyl enzyme rearranged into an alpha-beta unsaturated ester and inactivation was irreversible. In contrast to the situation prevailing with the Streptomyces albus G beta-lactamase, no turn-over of beta-iodopenicillanate was observed.     

Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

Haloenol lactones as inactivators and substrates of aldehyde dehydrogenase

Human aldehyde dehydrogenase (EC 1.2.1.3) isozymes E1 and E2 were irreversibly inactivated by stoichiometric concentrations of the haloenol lactones 3-isopropyl-6(E)-bromomethylene tetrahydro-pyran-2-one and 3-phenyl-6(E)-bromomethylene tetrahydro-pyran-2-one. No inactivation occurred with the corresponding nonhalogenated enol lactones. Both the dehydrogenase and esterase activities were abolished. Activity was not regained on dialysis or treatment with 2-mercaptoethanol. The inactivation was subject to substrate protection: NAD afforded protection which increased in the presence of the aldehyde-substrate competitive inhibitor chloral. Saturation kinetics gave positivey-axis intercepts, allowing the determination of binding constants. Inactivation stiochiometry determined with¹⁴C-labeled 3-(1-naphthyl)-6(E)-iodomethylene tetrahydropyran-2-one was found to correspond to the active-site number. The nonhalogenated lactone, 3-(1-naphthyl)-6(E)-methylene tetrahydropyran-1-one was shown to be a substrate for aldehyde dehydrogenase via its esterase function. Inactivation and enzymatic hydrolysis occurred within a similar time frame. Opening of the lactone ring to form enzyme-acyl intermediate with active site cysteine appears to be a necessary prerequisite to inactivation, since halogen in the lactone ring is nonreactive. Thus, the inactivation of aldehyde dehydrogenase by haloenol lactones is mechanism-based. Inactivation by haloenol lactones occurs in a manner analogous to that of chymotrypsin with which aldehyde dehydrogenase shares esterase activity and binding of haloenol lactones at the active site.     

Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.     

Functionally important residues of glutamic acid in E. coli pyrophosphatase. I. Chemical modification and localization in the primary structure]

Inorganic pyrophosphatase of E. coli is rapidly and irreversibly inactivated by 5-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K). The appearance in the absorption spectrum of a maximum at 340 nm testifies to the formation of an enzyme enol ester with the inhibitor. The non-hydrolyzable substrate analog CaPP1 partly protects the enzyme from inactivation. A peptide has been isolated from a tryptic hydrolysate of inactivated enzyme which contains an amino acid residue whose modification is critical for the enzyme activity. This peptide corresponds to residues 95-104 of pyrophosphatase and contains four dicarboxylic acid residues. A peptide containing a modified glutamic acid residue was isolated from modified pyrophosphatase hydrolyzed by protease v8. This peptide represents a fragment of a tryptic modified peptide and has a Glu-Ala-Gly-Glu (residues 98-1C1) structure. It is concluded that inactivation of E. coli pyrophosphatase by Woodward's reagent K is a result of selective modification of Glu98, apparently by the most reactive dicarboxylic amino acid within the enzyme active center.     

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Syntheses and kinetic evaluation of racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids as irreversible inactivators for carboxypeptidase A

Racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids were synthesized and evaluated as inactivators for carboxypeptidase A, a representative zinc-containing proteolytic enzyme. Only the threo-form of the inactivator is effective and its potency in terms of k(inact)/K(I) value is lower by 42-fold compared with 2-benzyl-3,4-epoxybutanoic acid, indicating that the alpha-methyl group affects adversely in the inactivation contrary to the expectation that it would enhance the inactivation activity of the inhibitor through additional interactions of the methyl group with a small cavity (alpha-methyl hole) present next to the S1' hydrophobic pocket. Of the enantiomeric pair, the inactivator having the (2S,3R)-configuration is more potent than its enantiomer by 44-fold. The observed kinetic results may be rationalized on the basis that the methyl group in the inactivator having the (2R,3S)-configuration experiences the van der Waals repulsive interactions with the bottom of the active site crevice in binding to CPA, casting a doubt on the presence of the so-called alpha-methyl hole at the active site of carboxypeptidase A.     

Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines

Quorum sensing, the ability of bacteria to sense their own population density through the synthesis and detection of small molecule signals, has received a great deal of attention in recent years. Acyl homoserine lactones (AHLs) are a major class of quorum sensing signaling molecules. In nature, some bacteria that do not synthesize AHLs themselves have developed the ability to degrade these compounds by cleaving the amide bond or the lactone ring. By inactivating this signal used by competing bacteria, the degrading microbe is believed to gain a competitive advantage. In this work we report that CYP102A1, a widely studied cytochrome P450 from Bacillus megaterium, is capable of very efficient oxidation of AHLs and their lactonolysis products acyl homoserines. The previously known substrates for this enzyme, fatty acids, can also be formed in nature by hydrolysis of the amide of AHLs, so CYP102A1 is capable of inactivating the active parent compound and the products of both known pathways for AHL inactivation observed in nature. AHL oxidation primarily takes place at the omega-1, omega-2, and omega-3 carbons of the acyl chain, similar to this enzyme's well-known activity on fatty acids. Acyl homoserines and their lactones are better substrates for CYP102A1 than fatty acids. Bioassay of the quorum sensing activity of oxidation products reveals that the subterminally hydroxylated AHLs exhibit quorum sensing activity, but are 18-fold less active than the parent compound. In vivo, B. megaterium inactivates AHLs by a CYP102A1 dependent mechanism that must involve additional components that further sequester or metabolize the products, eliminating their quorum sensing activity. Cytochrome P450 oxidation of AHLs represents an important new mechanism of quorum quenching.     

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.