δ-(L-α-Aminoadipyl)-L-Cysteinyl-D-Valine Synthetase, the Multienzyme Integrating the Four Primary Reactions in β-Lactam Biosynthesis, as a Model Peptide Synthetase

Aharonowitz, Yair; Bergmeyer, J.; Cantoral, Jesús M.; Cohen, Gerald; Demain, Arnold L.; Fink, U.; Kinghorn, James R.; Kleinkauf, Horst; MacCabe, Andrew; Palissa, H.

doi:10.1038/nbt0793-807

Cited by 66 publications

(42 citation statements)

References 43 publications

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“…These results establish that the L. monocytogenes GshF protein functions as a multimodular enzyme system that has within a single polypeptide the catalytic sites required for substrate activation (adenylation) and peptide bond formation. In these respects, the GshF protein superficially resembles the multifunctional NRPS, such as L-␣-aminoadipoyl-L-cysteine-D-valine (ACV) synthetase (2,6,20), that carry out stepwise assembly of small peptides on a single protein template. However, further characterization of the GshF-dependent reaction suggested that mechanistically it mimics the GshA-GshB two-enzyme system.…”

Section: Resultsmentioning

confidence: 99%

A Multidomain Fusion Protein inListeria monocytogenesCatalyzes the Two Primary Activities for Glutathione Biosynthesis

et al. 2005

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Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, ␥-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate ␥-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a ␥-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.Glutathione (␥-glutamyl-cysteinyl-glycine) (GSH) is the predominant low-molecular-weight peptide thiol present in living organisms. In bacteria it plays a pivotal role in many metabolic processes, chief among which are thiol redox homeostasis, protection against reactive oxygen species, protein folding, and provision of electrons via NADPH to reductive enzymes, such as ribonucleotide reductase. Low-molecular-weight nonribosomal peptides are assembled by the action of versatile multimodular enzymes termed nonribosomal peptide synthetases (NRPS) (22, 37) or through the consecutive actions of individual enzymes. GSH synthesis is a prime example of the latter, and GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (21). The ␥-carboxyl group of L-glutamate and the amino group of L-cysteine are ligated by the enzyme ␥-glutamylcysteine ligase (encoded by gshA) to give ␥-glutamylcysteine, which is then condensed with glycine in a reaction catalyzed by glutathione synthetase (encoded by gshB) to form GSH. Most grampositive bacteria do not contain GSH (9). However, a broad survey of the distribution of thiols in microorganisms revealed that several species of gram-positive bacteria, including Listeria, streptococci, and enterococci, produce significant amounts of GSH (23). The source of GSH in these bacteria has remained a puzzle, since their genomes do not contain a canonical gshB gene. The recent paper of Copley and Dhillon provides a clue to the origin of this GSH (7). These authors identified in the genomes of Listeria monocytogenes, Listeria innocua, Clostridium perfringens, and Pasteurella multocida an open reading frame (ORF) that is predicted to co...

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Section: Resultsmentioning

confidence: 99%

A Multidomain Fusion Protein inListeria monocytogenesCatalyzes the Two Primary Activities for Glutathione Biosynthesis

et al. 2005

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“…4A). Homology within CmaA to these enzymes corresponded to the functional domains of 500 to 600 amino acids which are conserved in the family of amino acid-activating enzymes (1,32). CmaA showed the highest degree of homology to functional domains in surfactin synthetase (10) and gramicidin synthetase GrsB (57), which specifically activate the branchedchain amino acids valine and leucine, respectively.…”

Section: Resultsmentioning

confidence: 99%

The biosynthetic gene cluster for coronamic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases

Ullrich

Bender

1994

J Bacteriol

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Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isoleucine, functions as an intermediate in the biosynthesis of coronatine, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. The DNA required for CMA biosynthesis (6.9 kb) was sequenced, revealing three distinct open reading frames (ORFs) which share a common orientation for transcription. The deduced amino acid sequence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-activating enzymes, including nonribosomal peptide synthetases. Furthermore, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of some nonheme iron(ll) enzymes which catalyze oxidative cyclizations.The deduced amino acid sequence of a 1.2-kb ORF designated cmaT was related to thioesterases of both procaryotic and eucaryotic origins. These data suggest that CMA assembly is similar to the thiotemplate mechanism of nonribosomal peptide synthesis. No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found. The start sites of two transcripts required for CMA biosynthesis were identified in the present study. pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts. Data obtained in the present study indicate that CMA biosynthesis is regulated at the transcriptional level by temperature.Coronatine (COR) is a chlorosis-inducing, non-host-specific phytotoxin produced by several Pseudomonas syringae pathovars and functions as an important virulence factor in the diseases incited by these bacteria (36,37,64). COR induces a number of responses in plants which can be reproduced by ethylene or indoleacetic acid, suggesting that the toxin alters host metabolism in a manner analogous to that of plant growth hormones (13,24,49). Recently, striking structural and functional homologies between COR, methyl jasmonate, and 12-oxo-phytodienoic acid were found, suggesting that COR mimics the octadecanoid signalling molecules of higher plants (15,19,63).COR consists of two distinct moieties, a polyketide component, coronafacic acid (CFA), and a cyclized amino acid, coronamic acid (CMA) (2-ethyl-1-aminocyclopropane 1-carboxylic acid). These are derived from separate biosynthetic pathways and coupled via amide bond formation ( Fig. 1) (42). Both moieties were previously shown to be required for the phytotoxic effects of COR (53, 63).The genes for COR biosynthesis in P. syringae pv. glycinea PG4180 are encoded within a 30-kb region of a 90-kb plasmid designated p4180A (67). Tn5 mutagenesis, phenotypic characterization, and feeding studies with exogenous CFA and CMA resulted in the recovery of mutants blocked in distinct biosynthetic steps (4). Genetic complementation studies with CMAdefective mutants and expression of CMA synthesis in another bacterium were used to localize a DNA...

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“…One encodes a 25-kDa protein (srfAORF4 (5) or the grsT product (13)) homologous to fatty acid synthase thioesterase type II. The other one, present in all peptide synthetases characterized so far, lies downstream from the sequence of the last amino acid binding domain and is homologous to fatty acid synthase thioesterase type I (6,7,14). We have shown that insertional inactivation or deletion of the thioesterase type I region results in a stable but unproductive surfactin synthetase (15), whereas deletion of srfAORF4 has no effect on peptide production (5).…”

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confidence: 91%

Engineering of Peptide Synthetases

Ferra¹,

Rodriguez²,

Tortora³

et al. 1997

Journal of Biological Chemistry

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Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.Two mechanisms for the biosynthesis of peptides are known to exist in bacteria and fungi, ribosomal synthesis and production by peptide synthetases. These are large enzymatic complexes responsible for the synthesis of hundreds of types of peptides, some of which have immunoactive, antibiotic, antifungal, or surfactant properties. Whereas polypeptides produced by ribosomal synthesis typically contain only the amino acids directly specified by the triplets of the genetic code, peptides built on synthetases often contain unusual amino or hydroxy acids as building blocks that are not present in proteins. The amino acids can be modified by peptide synthetases either through methylation, hydroxylation, or enantiomerization. The peptides are typically short (up to about 20 residues) and can be linear, circular, or branched (1, 2). Peptide synthesis proceeds by the "multiple carrier thiotemplate mechanism" (3, 4), and many of the details of these systems remain to be investigated experimentally. According to this mechanism each domain recognizes a specific amino (or hydroxy) acid that is covalently bound to the cofactor via a thioester bond after activation to the corresponding acyladenylate derivative. The growth of the polypeptide chain thus occurs through a series of thioester bond cleavages and the simultaneous formation of amide or ester bonds in the peptide. At the end of synthesis of each peptide, the chain is thought to be released from the enzyme by a thioesterase (TE) 1 activity encoded in the synthetase gene (1). Peptide synthetases are interesting not only from a scientific and evolutionary point of view but also for their biotechnological potential. Many enzymatically synthesized peptides are in fact biologically active, and some of them are industrially produced, among which are cyclosporins, surfactin, and fungicides. A growing number of laboratories are involved in applied research projects focused on the isolation and characterization of new peptide synthetases and on the genetic manipulation of peptide synthetase genes to optimize peptide production and to genetically modify the sequence of the peptides produced. In fact, the structural organization...

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δ-(L-α-Aminoadipyl)-L-Cysteinyl-D-Valine Synthetase, the Multienzyme Integrating the Four Primary Reactions in β-Lactam Biosynthesis, as a Model Peptide Synthetase

Cited by 66 publications

References 43 publications

A Multidomain Fusion Protein inListeria monocytogenesCatalyzes the Two Primary Activities for Glutathione Biosynthesis

A Multidomain Fusion Protein inListeria monocytogenesCatalyzes the Two Primary Activities for Glutathione Biosynthesis

The biosynthetic gene cluster for coronamic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases

Engineering of Peptide Synthetases

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