acyl-CoA Ligase Research Articles

Cloning, Sequencing, and Functional Analysis of the Biosynthetic Gene Cluster of Macrolactam Antibiotic Vicenistatin in

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning ∼64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well.

Cloning, sequencing, and functional analysis of the biosynthetic gene cluster of macrolactam antibiotic vicenistatin in Streptomyces halstedii.

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well.

Multiple Contributions from Long‐Chain Fatty Acid Metabolism in Y‐1 and MA‐10 Cells

Acute steroidogenesis in either Y-1 or MA-10 cells is sensitive to different effects of fatty acids compare to a chronic stimulation. A 3-h stimulation of StAR expression in both cell types was completely blocked by NDGA and AA861, each functioning as lipoxygenase inhibitors. However, the acute 15-min stimulation in Y-1 cells was inhibited by these agents by distinct mechanisms. The inhibition by NDGA was reversed by arachidonic, linoleic, and oleic acids. The inhibition by AA861 was insensitive to reversal by these acids. StAR expression was not affected by these short-term inhibitor treatments. These more rapid fatty acid reversible effects of NDGA are consistent with previously reported inhibition of mitochondrial Acyl CoA ligase. This may function in cooperation with StAR and PBR in providing fatty acid regulation of mitochondrial cholesterol transport. The acute effect of AA861 supports the involvement of an NDGA-insensitive lipoxygenase in the acute stimulation of mitochondrial cholesterol metabolism. The activity of MA-10 cells during prolonged treatments with cAMP appears to utilize each of these processes, which depend on different metabolism of fatty acids.

Diet-Induced Milk Fat Depression in Dairy Cows Results in Increased trans-10, cis-12 CLA in Milk Fat and Coordinate Suppression of mRNA Abundance for Mammary Enzymes Involved in Milk Fat Synthesis

Milk composition can be altered by diet, and one example is milk fat depression (MFD) in dairy cows. The biohydrogenation theory of MFD has implicated unique fatty acids formed by altered rumen biohydrogenation of PUFA; one example is trans-10, cis-12 conjugated linoleic acid (CLA). In the present study, we induced MFD with a high concentrate/low forage (HC/LF) diet and examined milk composition, milk fatty acid changes and mammary lipogenic mRNA abundance to determine the mechanism involved. The HC/LF diet reduced milk fat percentage by 25% and yield by 27% with no effect on dietary intake, milk production, protein or lactose. Milk fatty acids synthesized de novo in the mammary gland and fatty acids taken up from circulation were reduced to a similar extent (molar basis). MFD was also characterized by the appearance of trans-10, cis-12 CLA in the milk fat. We analyzed mammary mRNA abundance for lipogenic genes and detected reductions for acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), fatty acyl CoA ligase, glycerol phosphate acyltransferase (GPAT) and acylglycerol phosphate acyltransferase (AGPAT). There was no effect on the milk protein gene, kappa-casein. The reductions in mRNA were also correlated with the appearance of trans-10, cis-12 CLA in the milk fat for ACC, FAS, lipoprotein lipase and GPAT. This study demonstrates that diet-induced MFD involves coordinated effects on mRNA for mammary lipid synthesis pathways, and provides support for a mechanism involving alterations in transcriptional activation of these genes.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Localization of a non-syndromic X-linked mental retardation gene (MRX80) to Xq22-q24.

Isolated mental retardation is clinically and genetically heterogenous and may be inherited in an autosomal dominant, autosomal recessive, or X-linked manner. We report here a linkage analysis in a large family including 15 members, 6 of whom presenting X-linked non-syndromic mental retardation (MRX). Two-point linkage analysis using 23 polymorphic markers covering the entire X chromosome demonstrated significant linkage between the causative gene and DXS8055 with a maximum LOD score of 2.98 at theta = 0.00. Haplotype analysis indicated location for the disease gene in a 23.1 cM interval between DXS1106 and DXS8067. This MRX localization overlaps with 7 XLMR loci (MRX23, MRX27, MRX30, MRX35, MRX47, MRX53, and MRX63). This interval contains two genes associated with non-syndromic mental retardation (NSMR), namely the PAK3 gene, encoding a p21-activated kinase (MRX30 and MRX47) and the FACL4 gene encoding a fatty acyl-CoA ligase (MRX63). As skewed X-inactivation, an apparently constant feature in FACL4 carrier females was not observed in an obligate carrier belonging to the MRX family presented here, the PAK3 gene should be considered as the strongest candidate for this MRX locus.

A novel class of gene controlling virulence in plant pathogenic ascomycete fungi

Insertional mutants of the fungal maize pathogen Cochliobolus heterostrophus were screened for altered virulence. One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype. Analysis of sequence at the insertion site revealed a gene (CPS1) encoding a protein with two AMP-binding domains. CPS1 orthologs were detected in all Cochliobolus spp. examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants. Phylogenetic analysis suggested that CPS1 represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketidepeptide hybrids. Disruption of CPS1 caused reduced virulence of both race T and race O of C. heterostrophus on maize, of Cochliobolus victoriae on oats, and of Gibberella zeae on wheat. These results suggest that CPS1 functions as a general fungal virulence factor in plant pathogenic ascomycetes.

Deletion of a Single Amino Acid Residue from Different 4-Coumarate:CoA Ligases from Soybean Results in the Generation of New Substrate Specificities

Plant 4-coumarate:coenzyme A ligases, acyl-CoA ligases, peptide synthetases, and firefly luciferases are grouped in one family of AMP-binding proteins. These enzymes do not only use a common reaction mechanism for the activation of carboxylate substrates but are also very likely marked by a similar functional architecture. In soybean, four 4-coumarate:CoA ligases have been described that display different substrate utilization profiles. One of these (Gm4CL1) represented an isoform that was able to convert highly ring-substituted cinnamic acids. Using computer-based predictions of the conformation of Gm4CL1, a peptide motif was identified and experimentally verified to exert a critical influence on the selectivity toward differently ring-substituted cinnamate substrates. Furthermore, one unique amino acid residue present in the other isoenzymes of soybean was shown to be responsible for the incapability to accommodate highly substituted substrates. The deletion of this residue conferred the ability to activate sinapate and, in one case, also 3,4-dimethoxy cinnamate and was accompanied by a significantly better affinity for ferulate. The engineering of the substrate specificity of the critical enzymes that activate the common precursors of a variety of phenylpropanoid-derived secondary metabolites may offer a convenient tool for the generation of transgenic plants with desirably modified metabolite profiles.

The origin, diversity, and structure function relationships of insect luciferases.

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes.

Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases.

The synthesis of the catecholic siderophore bacillibactin is accomplished by the nonribosomal peptide synthetase (NRPS) encoded by the dhb operon. DhbE is responsible for the initial step in bacillibactin synthesis, the activation of the aryl acid 2,3-dihydroxybenzoate (DHB). The stand-alone adenylation (A) domain DhbE, the structure of which is presented here, exhibits greatest homology to other NRPS A-domains, acyl-CoA ligases and luciferases. It's structure is solved in three different states, without the ligands ATP and DHB (native state), with the product DHB-AMP (adenylate state) and with the hydrolyzed product AMP and DHB (hydrolyzed state). The 59.9-kDa protein folds into two domains, with the active site at the interface between them. In contrast to previous proposals of a major reorientation of the large and small domains on substrate binding, we observe only local structural rearrangements. The structure of the phosphate binding loop could be determined, a motif common to many adenylate-forming enzymes, as well as with bound DHB-adenylate and the hydrolyzed product DHB*AMP. Based on the structure and amino acid sequence alignments, an adapted specificity conferring code for aryl acid activating domains is proposed, allowing assignment of substrate specificity to gene products of previously unknown function.

Cryptosporidiumparvum: the first protist known to encode a putative polyketide synthase

We are reporting a putative multifunctional Type I polyketide synthase (PKS) gene from the apicomplexan Cryptosporidium parvum ( CpPKS1). The 40 kb intronless open reading frame (ORF) predicts a single polypeptide of 13,414 amino acids with a molecular mass of 1516.5 kDa. Sequence analysis identified at least 29 enzymatic domains within this protein. These domains are organized into an N-terminal loading unit, seven polyketide chain elongation modules, and a carboxy terminator unit. The loading domain consists of an acyl-CoA ligase (AL) and an acyl carrier protein (ACP). All seven elongation modules contain between two and five of the six domains required for the elongation of two-carbon (C2) acyl units, i.e. ketoacyl synthase, acyl transferase, dehydrase, enoyl reductase, ketoreductase and/or ACP. The carboxy terminator is homologous to various reductases, suggesting that the final elongated product is not hydrolytically released by thioesterases as observed in most Type I PKS and all fatty acid synthetase (FAS) systems, but by a reducing reaction, which has been demonstrated in some non-ribosomal peptide synthase systems. The protein sequence and domain organization of CpPKS1 protein resembles a previously reported C. parvum fatty acid synthase (CpFAS1), which is encoded by a 25 kb ORF. Maximum likelihood phylogenetic analysis of acyl transferases within PKS/FAS from C. parvum and other organisms clearly differentiates acetate-extending clades from those incorporating propionate. All acyl transferase domains from CpPKS1, and a previously reported CpFAS1, clustered within the acetate-extending group, suggesting the likelihood that only non-methylated C2 units are incorporated by C. parvum polyketide and fatty acid synthases. The expression of CpPKS1 was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence microscopy. Many polyketides are medically significant antibiotics, anticancer agents, toxins, or signaling molecules. Therefore, it is interesting to speculate what role CpPKS1 might play in this apicomplexan and the disease caused by this opportunistic infection of AIDS patients.

Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine.

Pyoverdines, the main siderophores of fluorescent pseudomonads, contain a peptide moiety, different for each pyoverdine, and an identical chromophore. While it has been shown that non-ribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of the peptide chain of pyoverdines, this was not demonstrated for the biosynthesis of the chromo-phore part. We found that PvsA, from Pseudomonas fluorescens ATCC 17400, and PvdL (PA2424), from Pseudomonas aeruginosa are similar NRPSs and functional homologues, necessary for the production of pyoverdine. Transcriptional lacZ fusions showed that pvdL is co-transcribed with the upstream PA2425 gene, encoding a putative thioesterase, and is iron-regulated via PvdS. Similarly, RT-PCR analysis revealed that expression of pvsA is repressed by iron. Analysis of the adenylation domains of PvsA, PvdL and their homologues, revealed that their N-terminus starts with an acyl-CoA ligase module, followed by three amino acid activation domains. Computer modelling of these domains suggests that PvsA in P. fluorescens and PvdL in P. aeruginosa are orthologues involved in the biosynthesis of the pyoverdine chromophore.

Techniques for Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl‐CoA:Amino Acid N‐Acyltransferase: The Amino Acid Conjugation Pathway

A wide variety of xenobiotic carboxylic acids are metabolized to their amino acid conjugates via a pathway that exists primarily in liver and kidney. This conjugation occurs in a two-step pathway catalyzed by two distinct types of enzymes, ligases and transferases. Measurements of acyl-CoA ligase activity include monitoring the rate of appearance of AMP or PPi, or the CoA adduct. N-acyltransferases catalyze formation of an amino acid conjugate from the CoA-activated intermediate, releasing CoA. This reaction is monitored by following the release of free CoA or the disappearance of the acyl-CoA adduct.

Expression of genes of lipid synthesis and altered lipid composition modulates L-glutamate efflux of Corynebacterium glutamicum.

L-Glutamate is made with Corynebacterium glutamicum on a scale of more than 106 tons/year. Nevertheless, formation of this amino acid is enigmatic and there is very limited molecular information available to unravel the apparently complex conditions leading to L-glutamate efflux. Here, we report the isolation and overexpression of the genes involved in lipid synthesis: acp, fadD 15, cma, cls, pgsA2, cdsA, gpsA, and plsC, and the inactivation of cma and cls. In addition, the consequences for phospholipid content, temperature sensitivity, as well as detergent-independent and detergent-dependent L-glutamate efflux were quantified. An in part strong alteration of the phospholipid composition was achieved; for instance, overexpression offadD15 encoding an acyl-CoA ligase resulted in an increase of phosphatidyl inositol from 12.6 to 30.2%. All strains, except that overexpressing acp (acyl carrier protein), exhibited increased temperature sensitivity, with the strongest sensitivity present upon cls (cardiolipin synthetase) inactivation. As a consequence of the genetically modified lipid synthesis, L-glutamate efflux changed quite dramatically; for instance, overexpression of plsC (acylglycerolacyl transferase) resulted in a detergent-triggered increase of L-glutamate accumulation from 92 mM to 108 mM, whereas acp overexpression reduced the accumulation to 24 mM. With some of the overexpressed genes, substantial L-glutamate excretion even without detergent addition was obtained when the fermentation temperature was elevated. These data show that the chemical and physical properties of the cytoplasmic membrane are altered and suggest that this is a necessary precondition to achieve L-glutamate efflux.

A fadD mutant of Sinorhizobium meliloti shows multicellular swarming migration and is impaired in nodulation efficiency on alfalfa roots.

Swarming is a form of bacterial translocation that involves cell differentiation and is characterized by a rapid and co-ordinated population migration across solid surfaces. We have isolated a Tn5 mutant of Sinorhizobium meliloti GR4 showing conditional swarming. Swarm cells from the mutant strain QS77 induced on semi-solid minimal medium in response to different signals are hyperflagellated and about twice as long as wild-type cells. Genetic and physiological characterization of the mutant strain indicates that QS77 is altered in a gene encoding a homologue of the FadD protein (long-chain fatty acyl-CoA ligase) of several microorganisms. Interestingly and similar to a less virulent Xanthomonas campestris fadD(rpfB) mutant, QS77 is impaired in establishing an association with its host plant. In trans expression of multicopy fadD restored growth on oleate, control of motility and the symbiotic phenotype of QS77, as well as acyl-CoA synthetase activity of an Escherichia coli fadD mutant. The S. meliloti QS77 strain shows a reduction in nod gene expression as well as a differential regulation of motility genes in response to environmental conditions. These data suggest that, in S. meliloti, fatty acid derivatives may act as intracellular signals controlling motility and symbiotic performance through gene expression.

Biosynthesis and degradation of glycerides in external mycelium of Glomus mosseae

The activities of enzymes involved in the glyceride metabolism of Glomus mosseae external mycelium are reported. Total mycelial homogenates were incubated with radiolabeled triolein and palmitic acid for various times under different conditions. The results obtained demonstrate the capacity of G. mosseae external mycelium to synthesize and hydrolyze its own acylglycerides. Neutral lipid biosynthesis progressively increased along with root colonization. Incorporation of [14C]-palmitate was mainly into triacylglycerols and as a minor fraction into diacylglycerols. The activity of palmitoyl-CoA ligase in external mycelium also increased in parallel with mycorrhiza development. The hydrolysis of triacylglycerols was very low at the beginning of colonization and then increased. However, lipase activity was lower than that of acyl-CoA ligase even at late stages of colonization. Thus, triacylglycerol biosynthesis apparently prevails over degradation during G. mosseae mycelium development in the period examined.

Analysis of two chromosomal regions adjacent to genes for a type II polyketide synthase involved in the biosynthesis of the antitumor polyketide mithramycin in Streptomyces argillaceus.

Mithramycin is an aromatic antitumour polyketide synthesized by Streptomyces argillaceus. Two chromosomal regions located upstream and downstream of the locus for the mithramycin type II polyketide synthase were cloned and sequenced. Analysis of the sequence revealed the presence of eight genes encoding three oxygenases (mtmOI, mtmOII and mtmOIII), three reductases (mtmTI, mtmTII and mtmTIII), a cyclase (mtm Y) and an acyl CoA ligase (mtmL). The three oxygenase genes were each inactivated by gene replacement. Inactivation of one of them (mtmOII) generated a non-producing mutant, while inactivation of the other two (mtmOl and mtmOIII) did not affect the biosynthesis of mithramycin. The mtmOII gene may code for an oxygenase responsible for the introduction of oxygen atoms at early steps in the biosynthesis of mithramycin leading to 4-demethylpremithramycinone. One of the reductases may be responsible for reductive cleavage of an intermediate from an enzyme and another for the reduction of a keto group in the side-chain of the mithramycin aglycon moiety. A hypothetical biosynthetic pathway showing in particular the involvement of oxygenase MtmOII and of various other gene products in mithramycin biosynthesis is proposed.

Localization of nervonic acid β-oxidation in human and rodent peroxisomes: impaired oxidation in Zellweger syndrome and X-linked adrenoleukodystrophy

Studies with purified subcellular organelles from rat liver indicate that nervonic acid (C24:1) is β-oxidized preferentially in peroxisomes. Lack of effect by etomoxir, inhibitor of mitochondrial β-oxidation, on β-oxidation of lignoceric acid (C24:0), a peroxisomal function, and that of nervonic acid (24:1) compared to the inhibition of palmitic acid (16:0) oxidation, a mitochondrial function, supports the conclusion that nervonic acid is oxidized in peroxisomes. Moreover, the oxidation of nervonic and lignoceric acids was deficient in fibroblasts from patients with defects in peroxisomal β-oxidation [Zellweger syndrome (ZS) and X-linked adrenoleukodystrophy (X-ALD)]. Similar to lignoceric acid, the activation and β-oxidation of nervonic acid was deficient in peroxisomes isolated from X-ALD fibroblasts. Transfection of X-ALD fibroblasts with human cDNA encoding for ALDP (X-ALD gene product) restored the oxidation of both nervonic and lignoceric acids, demonstrating that the same molecular defect may be responsible for the abnormality in the oxidation of nervonic as well as lignoceric acid. Moreover, immunoprecipitation of activities for acyl-CoA ligase for both lignoceric acid and nervonic acid indicate that saturated and monoenoic very long chain (VLC) fatty acids may be activated by the same enzyme. These results clearly demonstrate that similar to saturated VLC fatty acids (e.g., lignoceric acid), VLC monounsaturated fatty acids (e.g., nervonic acid) are oxidized preferentially in peroxisomes and that this activity is impaired in X-ALD. In view of the fact that the oxidation of unsaturated VLC fatty acids is defective in X-ALD patients, the efficacy of dietary monoene therapy, “Lorenzo's oil,” in X-ALD needs to be evaluated. —Sandhir, R., M. Khan, A. Chahal, and I. Singh. Localization of nervonic acid β-oxidation in human and rodent peroxisomes: impaired oxidation in Zellweger syndrome and X-linked adrenoleukodystrophy. J. Lipid Res. 1998. 39: 2161–2171.

Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.

The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of approximately 60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 A resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94 degrees occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the alpha-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.

A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule.

Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris (Xcc). The phenotype of mutants in one of the genes, rpfF, can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipoplysaccharide and lipid A from Xcc, and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.