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PubMed2003

Enzyme-assisted suicide: molecular basis for the antifungal activity of 5-hydroxy-4-oxonorvaline by potent inhibition of homoserine dehydrogenase.

The structure of the antifungal drug 5-hydroxy-4-oxonorvaline (HON) in complex with its target homoserine dehydrogenase (HSD) has been determined by X-ray diffraction to 2.6 A resolution. HON shows potent in vitro and in vivo activity against various fungal pathogens despite its weak (2 mM) affinity for HSD in the steady state. The structure together with structure-activity relationship studies, mass spectrometry experiments, and spectroscopic data reveals that the molecular mechanism of antifungal action conferred by HON involves enzyme-dependent formation of a covalent adduct between C4 of the nicotinamide ring of NAD(+) and C5 of HON. Furthermore, novel interactions are involved in stabilizing the (HON*NAD)-adduct, which are not observed in the enzyme's ternary complex structure. These findings clarify the apparent paradox of the potent antifungal actions of HON given its weak steady-state inhibition characteristics.

Aminolevulinic Acid↗
PubMed2006

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells.

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.

4-Butyrolactone↗
PubMed2005

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway.

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.

4-Butyrolactone↗
PubMed1987

Mechanism of renaturation of a large protein, aspartokinase-homoserine dehydrogenase.

The renaturation of aspartokinase-homoserine dehydrogenase and of some of its smaller fragments has been investigated after complete unfolding by 6 M guanidine hydrochloride. Fluorescence measurements show that a major folding reaction occurs rapidly (in less than a few seconds) after the protein has been transferred to native conditions and results in the shielding of the tryptophan residues from the aqueous solvent; this step also takes place in the fragments and probably corresponds to the independent folding of different segments along the polypeptide chain. The reappearance of the kinase activity, which is an index of the formation of "native" structure within a single chain, is much slower (a few minutes) and has the following properties: it is involved in a kinetic competition with the formation of aggregates; it has an activation energy of 22 +/- 5 kcal/mol; it is not related to a slow reaction in unfolding and thus probably not controlled by the cis-trans isomerization of X-Pro peptide bonds; its rate is inversely proportional to the solvent viscosity. It seems as if this reaction is limited by the mutual arrangement of the regions that have folded rapidly and independently. It is proposed that the mechanism where a fast folding of domains is followed by a slow pairing of folded domains could be generalized to other long chains composed of several domains; such a slow pairing of folded domains would correspond to a rate-limiting process specific to the renaturation of large proteins. The reappearance of the dehydrogenase activity measures the formation of a dimeric species. The dimerization can occur only after each chain has reached its "native" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartokinase Homoserine Dehydrogenase↗
PubMed1989

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity.

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.

Alcohol Oxidoreductases↗
PubMed1978

Threonine inhibition of the aspartokinase--homoserine dehydrogenase I of Escherichia coli. Threonine binding studies.

Both activities of the aspartokinase--homoserine I (AK-HSD) of Escherichia coli are inhibited by threonine. Careful threonine binding studies have now been done which have allowed us to distinguish the various effects of threonine on the enzyme. The ultrafiltration technique of H. Paulus ((1969) Anal. Biochem. 32, 101) for measuring ligand binding was shown to be comparable with equilibrium dialysis techniques. Reduction in error by utilization of this procedure enabled us to obtain evidence for two different sets of threonine sites by direct binding studies. The binding data were mathematically consistent with two independent classes of threonine sites, each of which contained four sites per tetramer and had a Hill coefficient of about 2.3--2.5. KD for the second set of sites was five- to tenfold greater than the high affinity sites, depending upon conditions. The data now suggest that the sequential model for site--site interactions adequately describes the cooperativity of threonine binding to the high affinity set of sites.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1975

Structure-function relationships in glucagon: properties of highly purified des-His-1-, monoiodo-, and (des-Asn-28, Thr-29)(homoserine lactone-27)-glucagon.

We have compared the ability of glucagon and three highly purified derivatives of the hormone to activate hepatic adenylate cyclase (an expression of biological activity of the hormone) and to compete with [125]glucagon for binding to sites specific for glucagon in hepatic plasma membranes. Relative to that of glucagon, biological activity and affinity of [des-Asn-28,Thr-29](homoserine lactone-27)-glucagon, prepared by CNBr treatment of glucagon, were reduced equally by 40- to 50-fold. By contrast, des-His-1-glucagon, prepared by an insoluble Edman reagent and highly purified (less than 0.5% contamination with native glucagon), displayed a 15-fold decrease in affinity but a 50-fold decrease in biological activity relative to that of the native hormone. At maximal stimulating concentrations, des-His-1-glucagon yielded 70% of the activity given by saturating concentrations of glucagon. Thus, des-His-1-glucagon can be classified as a partial weak agonist. Highly purified monoiodoglucagon and native glucagon displayed identical biological activity and affinity for the binding sites. Our findings suggest that the hydrophilic residues at the terminus of the carboxy region of glucagon are involved in the process of recognition at the glucagon receptor but do not participate in the sequence of events leading to activation of adenylate cyclase. The amino-terminal histidyl residue in glucagon plays an important but not obligatory role in the expression of hormone action and contributes to a significant extent in the recognition process.

Adenylyl Cyclases↗
PubMed1971

Homoserine and diaminobutyric acid in the mucopeptide-precursor-nucleotides and cell walls of some plant-pathogenic corynebacteria.

The nucleotide precursors of cell-wall mucopeptide were prepared by vancomycin inhibition of Corynebacterium insidiosum and Corynebacterium poinsettiae. The amino acid in the third position in the former peptide was shown by optical rotatory dispersion of the bisdinitrophenyl derivative to be l-diaminobutyric acid. Homoserine in the nucleotide from C. poinsettiae was catalytically oxidized to aspartic acid, which was then shown to be the l-isomer by optical rotatory dispersion of the trinitrophenyl compound. The sequence for C. insidiosum was UDP-N-acetylmuramyl-glycyl-isoglutamyl- (gamma-acetyl)-diaminobutyryl-alanyl-alanine. The cell walls of C. insidiosum and Corynebacterium sepedonicum contained diaminobutyric acid that was optically inactive. It is proposed, therefore, that the primary peptide chain contains the l-isomer with its gamma-amino group blocked by acetylation and that cross-linking is achieved by means of the d-isomer, analogous to d-ornithine in C. poinsettiae.

Acylation↗
PubMed2005

Homoserine and asparagine are host signals that trigger in planta expression of a pathogenesis gene in Nectria haematococca.

Some pathogenesis-related genes are expressed in fungi only when the pathogen is in the host, but the host signals that trigger these gene expressions have not been identified. Virulent Nectria haematococca infects pea plants and requires either pelA, which is induced by pectin, or pelD, which is induced only in planta. However, the host signal(s) that trigger pelD expression was unknown. Here we report the isolation of the host signals and identify homoserine and asparagine, two free amino acids found in uniquely high levels in pea seedlings, as the pelD-inducing signals. N. haematococca has evolved a mechanism to sense the host tissue environment by using the high levels of two free amino acids in this plant, thereby triggering the expression of pelD to assist the pathogenic process.

Asparagine↗
PubMed1984

Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12.

In Escherichia coli, AK I- HDH I and AK II- HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the common pathway leading to threonine and methionine. An extensive amino acid sequence comparison of both molecules reveals two main features on each of them: (i) two segments, each of about 130 amino acids, covering the first one-third of the polypeptide chain, are similar to each other and (ii) two segments, each of about 250 amino acids and covering the COOH-terminal 500 amino acids also present a significant homology. These findings suggest that these two regions may have evolved independently of each other by a process of gene duplication and fusion previous to the appearance of an ancestral aspartokinase-homoserine dehydrogenase molecule.

Amino Acid Sequence↗
PubMed2000

The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase.

Several different species of Pseudomonas: produce N:-acylhomoserine lactones (AHLs), quorum-sensing signal molecules which are involved in the cell-density-dependent control of secondary metabolite and virulence gene expression. When Pseudomonas fluorescens F113 was cross-streaked against AHL biosensors capable of sensitively detecting either short (C(4)-C(8)) or long (C(10)-C(14)) acyl chain AHLs, no activity was detectable. However, by extracting cell-free stationary-phase culture supernatants with dichloromethane followed by reverse-phase HPLC, three distinct fractions were obtained capable of activating the AHL biosensors. Three AHLs were subsequently characterized using high-resolution MS and chemical synthesis. These were (i) N:-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3OH, C(14:1)-HSL), a molecule previously known as the Rhizobium leguminosarum small bacteriocin as a consequence of its growth inhibitory properties, (ii) N:-decanoylhomoserine lactone (C(10)-HSL) and (iii) N:-hexanoylhomoserine lactone (C(6)-HSL). A gene (hdtS) capable of directing synthesis of all three P. fluorescens AHLs in Escherichia coli was cloned and sequenced. In vitro transcription/translation of hdtS yielded a protein of approximately 33 kDa capable of directing the synthesis of 3OH, C(14:1)-HSL, C(10)-HSL and C(6)-HSL in E. coli. HdtS does not belong to either of the known AHL synthase families (LuxI or LuxM) and is related to the lysophosphatidic acid acyltransferase family. HdtS may therefore constitute a member of a third protein family capable of AHL biosynthesis.

4-Butyrolactone↗
PubMed1994

Molecular genetics of the maize (Zea mays L.) aspartate kinase-homoserine dehydrogenase gene family.

Aspartate kinase (AK) and homoserine dehydrogenase (HSDH) are enzymes in the aspartate-derived amino acid biosynthetic pathway. Recent biochemical evidence indicates that an AK-HSDH bifunctional enzyme exists in maize (Zea mays L.). In this report, we characterize three genes that encode subunits of AK-HSDH. Two cDNAs, pAKHSDH1 and pAKHSDH2, containing the full-coding sequence, and one partial cDNA, pAKHSDH3, encode amino acid sequences similar to the reported monofunctional AK and HSDH enzymes from prokaryotes and yeast (Saccharomyces cerevisiae) and to AK-HSDH bifunctional enzymes of prokaryotes, yeast, carrot (Daucus carota), and Arabidopsis thaliana. Immunological and biochemical analyses verify that the cDNAs encode AK-HSDH and indicate that both the AK and HSDH activities are feedback inhibited by threonine. RNA blots identify a 3.2-kb transcript in all maize tissues examined. pAKHSDH1 and pAKHSDH2 map to chromosomes 4L and 2S, respectively. This study shows that maize contains AK-HSDH bifunctional enzyme(s) encoded by a small gene family of at least three genes. Maize AK-HSDH has conserved sequences found in communication modules of prokaryotic two-component regulatory systems, which has led us to propose that maize AK-HSDH may be involved in a similar regulatory mechanism.

Amino Acid Sequence↗
PubMed2005

The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones.

The transcriptional regulator MvfR is required for full Pseudomonas aeruginosa virulence, the function of multiple quorum sensing (QS)-regulated virulence factors and the synthesis of 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal (PQS). Here we investigate the role of MvfR in the QS circuitry and P. aeruginosa pathogenesis. We demonstrate using a combination of biochemical and molecular approaches, including transcription profiling, that MvfR is involved in the regulation of multiple P. aeruginosa QS-controlled genes without altering the expression of lasRI/rhlRI or the production of N-acyl-L-homoserine lactone (AHL) signals. Dissection of how mvfR is interwoven into the P. aeruginosa QS circuitry reveals that the MvfR system, through the essential contribution of PqsE, positively regulates a subset of genes dependant on both LasR and RhlR. Animal studies show that MvfR contributes to P. aeruginosa virulence by controlling the transcription of genes not under RhlR regulation, and that reduced virulence of a mvfR mutant is caused by the loss of pqsE expression and not only a deficiency in HAQs/PQS production. This study provides novel insights into the unique role of the MvfR system in AHL-mediated QS and further supports its importance in P. aeruginosa pathogenesis.

Animals↗
PubMed1977

Studies on the partial synthesis of protein analogs by direct coupling to terminal homoserine lactone derivatives. Experiments with basic pancreatic trypsin inhibitor.

Treatment of basic pancreatic trypsin inhibitor (BPTI, I) with cyanogen bromide smoothly yields the chain cleaved derivative II. The utility of the seco-lactone (II) in the partial synthesis of protein analogues has been investigated. It is shown that the homoserine lactone ring is sufficiently reactive to combine directly with the radiolabelled synthetic peptide glycylglycylanine t-butyl ester in both aqueous and non-aqueous solution, leading to a BPTI analogue which has been purified and characterized.

Cyanogen Bromide↗
PubMed1978

Homoserine dehydrogenase of Rhodospirillum rubrum. Physical and chemical characterization.

A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110000 and consists of two subunits of identical molecular weight of 55000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220000. Titrations of the native and detergent-treated enzyme with a variety of sulfhydryl reagents show 2 mol free--SH groups per 110000 g, one of which is buried in the protein interior. L-Threonine and/or high concentrations of salt can expose the buried--SH group, and this--SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by L-threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.

Alcohol Oxidoreductases↗
PubMed2002

Characterization of N-acyl homoserine lactone overproducing mutants of Burkholderia multivorans ATCC 17616.

Burkholderia multivorans ATCC 17616 ordinarily produces insufficient amounts of N-acyl homoserine lactones (AHLs) to promote AHL-dependent formation of the pigment violacein by the reporter strain Chromobacterium violaceum CV026. We have isolated AHL-overproducing mutants of strain 17616 by screening for variants which do cross-feed AHLs to strain CV026. Nucleotide-sequence analysis of the bmuIR locus which specifies AHL synthase (BmuI) and AHL-binding transcriptional activator protein (BmuR) indicated that the increased capacity to produce AHLs was not a consequence of changes upstream or internal to the bmuI or bmuR genes. We conclude that the mutations leading to AHL overproduction lie outside the bmuI/bmuR locus.

4-Butyrolactone↗
PubMed1982

Regulation of cephamycin C synthesis, aspartokinase, dihydrodipicolinic acid synthetase, and homoserine dehydrogenase by aspartic acid family amino acids in Streptomyces clavuligerus.

The effect of the cephalosporin precursors and amino acids of the aspartic acid family on antibiotic production by Streptomyces clavuligerus was investigated DL-meso-Diaminopimelate and L-lysine each stimulated specific antibiotic production by 75%. A fourfold increase in specific production was obtained by simultaneous addition of the two compounds. The stimulation could be further increased by adding valine to the two effectors. In the streptomycetes the alpha-aminoadipyl side chain of the cephalosporin antibiotics is derived from lysine. Streptomycetes, like other bacteria, are expected to produce lysine from aspartic acid; therefore, the feedback control mechanisms operating in the aspartic acid family pathway of S. clavuligerus, which may affect the flow of carbon to alpha-aminoadipic acid, were investigated. Threonine inhibited antibiotic production by 41% when added to minimal medium at a concentration of 10 mM. Simultaneous addition of 10 mM lysine completely reversed this inhibition. The aspartokinase of S. clavuligerus was found to be subject to concerted feedback inhibition by threonine and lysine. Threonine may act to limit the supply of lysine available for cephamycin C biosynthesis via this concerted mechanism. Single or simultaneous addition of any other amino acid of the aspartate family in the in vitro assay did not inhibit aspartokinase activity. Activity was stimulated by lysine. Aspartokinase biosynthesis was partially repressed by methionine or isoleucine at concentrations higher than 10 mM. Methionine, but not isoleucine, inhibited cephamycin C synthesis by 27% when added to minimal medium at a concentration of 10 mM. Dihydrodipicolinate synthetase, the first specific enzyme of the lysine branch, was not inhibited by lysine but was partially inhibited by high concentrations of 2,6-diaminopimelate and alpha-aminoadipate; it was slightly repressed by diaminopimelic acid. Homoserine dehydrogenase activity was inhibited by threonine and partially repressed by isoleucine. It appears that S. clavuligerus aspartokinase is a key step in the control of carbon flow toward alpha-aminoadipic acid.

2-Aminoadipic Acid↗
PubMed2004

Azithromycin inhibits MUC5AC production induced by the Pseudomonas aeruginosa autoinducer N-(3-Oxododecanoyl) homoserine lactone in NCI-H292 Cells.

The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus. Pseudomonas aeruginosa is one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease. The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C(12)-HSL)] not only regulates bacterial virulence but also is associated with the immune response. In this study, we investigated whether 3O-C(12)-HSL could stimulate the production of a major mucin core protein, MUC5AC. The effect of a macrolide on MUC5AC production was also studied. 3O-C(12)-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners. A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C(12)-HSL. 3O-C(12)-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-kappa B phosphorylation in cells, and this induction was suppressed by azithromycin. 3O-C(12)-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059. Our findings suggest that the P. aeruginosa autoinducer 3O-C(12)-HSL contributes to excessive mucin production in chronic bacterial infection. Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.

4-Butyrolactone↗