Synthesis of an actinomycin-related peptide lactone from the corresponding cyclic pentapeptide by N,O-acyl shift

The N,O-acyl shift was investigated as a method for the synthesis of an O-peptide or peptide lactone from a linear or cyclic peptide respectively. Protected derivatives of glycyl-L-threonine could be converted to O-peptides by the action of HCl/dioxane at room temperature and N-acylated under conditions which precluded a reverse O,N-acyl shift. For effecting N,O-acyl shift in cyclo(Thr-D-Val-Pro-Sar-MeAla) this reagent was unsatisfactory and p-toluenesulfonic acid in dioxane at 80 degrees was used instead. The resulting crystalline peptide lactone p-toluenesulfonate salt was N-acylated with 3-benzyloxy-4-methyl-2-nitrobenzoyl chloride to afford a known intermediate in the synthesis of 5,5'-MeAla actinomycin D. This approach constitutes a novel synthetic route to actinomycins and potentially to other peptide lactone antibiotics.     

Crystal structures of a quorum-quenching antibody

A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.     

The plant pathogen Erwinia amylovora produces acyl-homoserine lactone signal molecules in vitro and in planta

We report for the first time the production of acyl homoserine lactones (AHLs) by Erwina amylovora, an important quarantine bacterial pathogen that causes fire blight in plants. E. amylovora produces one N-acyl homoserine lactone [a N-(3-oxo-hexanoyl)-homoserine lactone or a N-(3-hydroxy-hexanoyl)-homoserine lactone] quorum sensing signal molecule both in vitro and in planta (pear plant). Given the involvement of AHLs in plant pathogenesis, we speculate that AHL-dependent quorum sensing could play an important role in the regulation of E. amylovora virulence.     

Oryza sativa rice plants contain molecules that activate different quorum-sensing N-acyl homoserine lactone biosensors and are sensitive to the specific AiiA lactonase

Gram-negative bacteria most often use N-acyl homoserine lactones (AHLs) as intercellular quorum-sensing signal molecules. In this study, it was demonstrated that rice plants contain AHL mimic molecules that are very sensitive to the highly specific AiiA lactonase enzyme and can activate three different AHL bacterial biosensors, indicating that the compounds have a homoserine lactone structure and could be AHLs. The possible source and biological significance of this finding are discussed.     

Bacterial populations in the rhizosphere of tobacco plants producing the quorum-sensing signals hexanoyl-homoserine lactone and 3-oxo-hexanoyl-homoserine lactone

A tobacco line genetically modified to produce two N-acyl homoserine lactones and its non-transformed parental line were grown in non-sterile soil. Microbial populations inhabiting the bulk soil, and those colonizing the root system of the two tobacco lines, were analyzed using cultivation-independent (phospholipid fatty acid and denaturing gradient gel electrophoresis) and cultivation-based assays. The cell density of total cultivable bacteria, fluorescent pseudomonads, sporulated, and thermotolerant bacteria was also determined in a time-course experiment (15 weeks). A possible "rhizosphere effect" related to the development of the plant was seen. However, no dissimilarities in cell population densities or population ratios of the microbial groups were detected in the rhizosphere of the two plant lines. Similarly, bacterial communities that either produced N-acyl homoserine lactone or degraded the signal hexanoyl homoserine lactone were enumerated from the two plant lines. No noticeable differences were evidenced from one plant genotype to the other. Whilst the transgenic plants released detectable amounts of the quorum-sensing signal molecules and efficiently cross-talked with the surrounding microbial populations, the bias generated by these signals in the reported experimental conditions therefore appears to remain weak, if not non-existent.     

Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.     

Sensitive whole-cell biosensor suitable for detecting a variety of N-acyl homoserine lactones in intact rhizosphere microbial communities

To investigate quorum sensing in rhizosphere soil, a whole-cell biosensor, Agrobacterium tumefaciens(pAHL-Ice), was constructed. The biosensor responded to all N-acyl homoserine lactones (AHLs) tested, except C(4) homoserine lactone, with a minimum detection limit of 10(-12) M, as well as to both exogenously added AHLs and AHL-producing bacterial strains in soil. This highly sensitive biosensor reveals for the first time the increased AHL availability in intact rhizosphere microbial communities compared to that in bulk soil.     

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.     

假单胞菌合成环脂肽转录调控的研究进展

假单胞菌所合成的环脂肽是一类由环状的寡肽连接一个脂肪酸链组成的两亲性分子,利用巯基化模块由非核糖体肽合成酶合成。环脂肽的生物合成受到严格、复杂的调控,GacS/GacA双组分系统和群体感应系统是其中两类重要的调控系统。本文总结假单胞菌合成环脂肽的调控机制及相关调控因子;对基于PCR的高通量分子筛选方法获取特定环脂肽进行分析,同时对基于调控机制的遗传改造提高假单胞菌产环脂肽的能力和获取更多新型环脂肽等方面的应用进行 展望。     

Conformational analysis of peptide T and of its C-pentapeptide fragment

The synthetic peptide of sequence H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, termed peptide T, a competitor of the Human Immunodeficiency Virus in the binding to human T cells, and its C-terminal pentapeptide fragment, were studied by 1H-nmr in DMSO solution to determine conformational preferences. The observation of nuclear Overhauser enhancements (NOEs) for both peptides, and unusual finding for small linear peptides, allowed complete sequence-specific resonance assignments. Long-range NOEs, ring-current shifts, and the very small temperature coefficient of the Thr8 NH chemical shift suggest, for the zwitterionic form of peptide T, the presence in solution of a beta-turn involving Thr5, Asn6, Tyr7 and Thr8. This conformational feature is consistent with previous structure-activity relationship studies indicating the invariance of the same residues in several potent pentapeptide analogues. The studied pentapeptide fragment, although less structured, shows some tendency to fold even in a polar solvent such as DMSO. Preliminary chemotaxis data on some pentapeptide analogues are consistent with our structural model.     

Orthogonal ligation strategies for peptide and protein

This review focuses on the concept, criteria, and methods of an orthogonal amide ligating strategy suitable for syntheses of peptides, peptide mimetics, and proteins. Utilizing unprotected peptides or proteins derived from chemical or biosynthetic sources, this ligation strategy has been shown to be general and exceptionally mild. Its orthogonality in ligating two unprotected segments with free N-terminal (NT)-amines at a specific NT-amine is achieved through a chemoselective capture step and then an intramolecular acyl transfer reaction. Both coupling reagents for enthalpic activation and protection schemes therefore become unnecessary. More than a dozen orthogonal ligation methods based on either imine or thioester captures have been developed to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. Because unprotected peptides and proteins of different sizes and forms can be obtained from either chemical or recombinant sources, orthogonal ligation removes the size limitation imposed on the chemical synthesis of a protein with a native or non-native structure. Furthermore, by using building blocks from biosynthetic sources, orthogonal ligation provides a unifying operational concept for both total and semisynthesis of peptides and proteins.     

Catabolic pathway of gamma-caprolactone in the biocontrol agent Rhodococcus erythropolis

Gamma-caprolactone (GCL) is well-known as a food flavor and has been recently described as a biostimulant molecule promoting the growth of bacteria with biocontrol activity against soft-rot pathogens. Among these biocontrol agents, Rhodococcus erythropolis, characterized by a remarkable metabolic versatility, assimilates various γ-butyrolactone molecules with a branched-aliphatic chain, such as GCL. The assimilative pathway of GCL in R. erythropolis was investigated by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. This analysis suggests the involvement of the lactonase QsdA in ring-opening, a feature confirmed by heterologous expression in Escherichia coli. According to proteome analysis, the open-chain form of GCL was degraded by β- and ω-oxidation coupled to the Krebs cycle and β-ketoadipate pathway. Ubiquity of qsdA gene among environmental R. erythropolis isolates was verified by PCR. In addition to a previous N-acyl homoserine lactone catabolic function, QsdA may therefore be involved in an intermediate degradative step of cyclic recalcitrant molecules or in synthesis of flavoring lactones.     

Effector-stimulated single molecule protein-DNA interactions of a quorum-sensing system in Sinorhizobium meliloti

Intercellular communication by means of small signal molecules coordinates gene expression among bacteria. This population density-dependent regulation is known as quorum sensing. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti Rm1021 possesses the Sin quorum sensing system based on N-acyl homoserine lactones (AHL) as signal molecules. Here, we demonstrate that the LuxR-type regulator ExpR binds specifically to a target sequence in the sinRI locus in the presence of different AHLs with acyl side chains from 8 to 20 carbons. Dynamic force spectroscopy based on the atomic force microscope provided detailed information about the molecular mechanism of binding upon activation by six different AHLs. These single molecule experiments revealed that the mean lifetime of the bound protein-DNA complex varies depending on the specific effector molecule. The small differences between individual AHLs also had a pronounced influence on the structure of protein-DNA interaction: The reaction length of dissociation varied from 2.6 to 5.8 A. In addition, dynamic force spectroscopy experiments indicate that N-heptanoyl-DL-homoserine lactone binds to ExpR but is not able to stimulate protein-DNA interaction.     

Organocatalytic α-hydroxymethylation of cyclopentanone with aqueous formaldehyde: Easy access to chiral δ-lactones

Optically active lactones are important synthons in perfume and aroma manufacturing. Therefore, developments of efficient asymmetric syntheses are desired. Organocatalytic asymmetric α-hydroxymethylations of cyclopentanone with aqueous formaldehyde have been developed, to furnish the corresponding α-(hydroxymethyl)cyclopentanone with high enantioselectivity. Further chemical transformation of α-(hydroxymethyl)cyclopentanone gave the key intermediate for jasmine lactone, which is widely found in fruits and flowers.     

Development of the enzyme-catalysed Baeyer–Villiger reaction as a useful technique in organic synthesis

The current practices of using monooxygenase enzymes to perform regio- and stereoselective oxidation reactions in organic syntheses are reviewed. The isolation of a monooxygenase from Pseudomonas putida NCIMB 10007 and its use in the conversion of bicyclo[3.2.0]hept-2-en-6-one into two isomeric optically active lactones is described. The monooxygenase utilises NADH as cofactor and NADH-recycling is accomplished using formate and formate dehydrogenase. As alternative methodology, it is shown that a secondary alcohol can be converted into a chiral lactone using a dehydrogenase and a monooxygenase working in tandem with in situ cofactor recycling. © 1993 Wiley-Liss, Inc.     

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.     

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998