Investigations of the biosynthesis of the phytotoxin coronatine

1994 ◽  
Vol 72 (1) ◽  
pp. 86-99 ◽  
Author(s):  
Ronald J. Parry ◽  
Sunil V. Mhaskar ◽  
Ming-Teh Lin ◽  
Alan E. Walker ◽  
Robson Mafoti
Keyword(s):  
Amino Acid ◽  
Pseudomonas Syringae ◽  
Cyclopropane Ring ◽  
Carbonyl Oxygen ◽  
Oxidative Cyclization ◽  
Amide Bond ◽  
Hydrogen Atoms ◽  
Coronafacic Acid ◽  
Polyketide Chain ◽  
Oxygen Atoms

The biosynthesis of the phytotoxin coronatine has been investigated by administration of isotopically labeled precursors to Pseudomonas syringae pv. glycinea. The structure of coronatine contains two moieties of distinct biosynthetic origin, a bicyclic, hydrindanone carboxylic acid (coronafacic acid) and a cyclopropyl α-amino acid (coronamic acid). Investigations of coronafacic acid biosynthesis have shown that this compound is a polyketide derived from three acetate units, one butyrate unit, and one pyruvate unit. The two carbonyl oxygen atoms of coronafacic acid were found to be derived from the oxygen atoms of acetate. Additional experiments are described that rule out some possible modes for assembly of the polyketide chain. Coronamic acid is shown to be derived from L-isoleucine via the intermediacy of L-alloisoleucine. Examination of the mechanism of the cyclization of L-alloisoleucine to coronamic acid revealed that the formation of the cyclopropane ring takes place with the removal of only two hydrogen atoms from the amino acid, one at C-2 and the other at C-6. The nitrogen atom at C-2 of L-alloisoleucine is shown to be retained. On the basis of these observations, a mechanism is postulated for the cyclization reaction that involves the diversion of an enzymatic hydroxylation reaction into an oxidative cyclization. Finally, a precursor incorporation experiment with deuterium-labeled coronamic acid demonstrated that free coronamic acid can be efficiently incorporated into coronatine. This observation indicates that the cyclization of L-alloisoleucine to coronamic acid can occur before formation of the amide bond between coronafacic acid and coronamic acid.

Use of Tn5-gusA5to investigate environmental and nutritional effects on gene expression in the coronatine biosynthetic gene cluster ofPseudomonas syringaepv.glycinea

1997 ◽  
Vol 43 (6) ◽  
pp. 517-525 ◽  
Author(s):  
David A. Palmer ◽  
Carol L. Bender ◽  
Shashi B. Sharma
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Nitrogen Sources ◽  
Biosynthetic Gene Cluster ◽  
Amide Bond ◽  
Transcriptional Level ◽  
Coronafacic Acid ◽  
Genes Encoding

Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafacic acid (CFA) coupled via an amide bond to the ethylcyclopropyl amino acid coronamic acid (CMA). Both CFA and CMA function as intermediates in the pathway to coronatine, and genes encoding their synthesis have been localized; however, the precise factors that regulate the production of COR and its precursors remain unclear. In the present study, a λ delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster. Selected carbon (fructose and xylose) and amino acid (isoleucine and valine) sources significantly decreased COR biosynthesis at the transcriptional level. Transcriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18–24 °C and significantly less expression at 14 and 30 °C. Interestingly, changes in osmolarity and the addition of complex carbon and nitrogen sources to the growth medium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced. These results indicate that multiple factors impact COR production and only some of these directly affect transcription in the COR gene cluster.Key words: transcriptional fusion, glucuronidase, gene expression, reporter gene.

scholarly journals Characterization of CmaA, an Adenylation-Thiolation Didomain Enzyme Involved in the Biosynthesis of Coronatine

2004 ◽  
Vol 186 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Robin Couch ◽  
Sarah E. O'Connor ◽  
Heather Seidle ◽  
Christopher T. Walsh ◽  
Ronald Parry
Keyword(s):  
Amino Acid ◽  
Carboxylic Acid ◽  
Pseudomonas Syringae ◽  
Cyclopropane Ring ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Biosynthetic Gene Cluster ◽  
Amide Bond ◽  
T Domain

ABSTRACT Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-32PPi exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain l-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for l-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4′-phosphopantetheinyl group, reacts with the AMP derivative of l-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and l-[G-3H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the l-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.

scholarly journals Coronafacoyl Phytotoxin Biosynthesis and Evolution in the Common Scab Pathogen Streptomyces scabiei

2017 ◽  
Vol 83 (19) ◽  
Author(s):  
Luke Bown ◽  
Yuting Li ◽  
Fabrice Berrué ◽  
Joost T. P. Verhoeven ◽  
Suzanne C. Dufour ◽  
...  
Keyword(s):  
Amino Acid ◽  
Pseudomonas Syringae ◽  
Pathogenic Bacteria ◽  
Amide Bond ◽  
Supporting Evidence ◽  
Biosynthetic Pathways ◽  
Biosynthetic Genes ◽  
Phytopathogenic Bacteria ◽  
Content Type ◽  
Streptomyces Scabiei

ABSTRACT Coronafacoyl phytotoxins are an important family of plant toxins that are produced by several different phytopathogenic bacteria, including the gammaproteobacterium Pseudomonas syringae and the actinobacterium Streptomyces scabiei (formerly Streptomyces scabies). The phytotoxins consist of coronafacic acid (CFA) linked via an amide bond to different amino acids or amino acid derivatives. Previous work suggested that S. scabiei and P. syringae use distinct biosynthetic pathways for producing CFA, which is subsequently linked to its amino acid partner to form the complete phytotoxin. Here, we provide further evidence that the S. scabiei CFA biosynthetic pathway is novel by characterizing the role of CYP107AK1, a predicted cytochrome P450 that has no homologue in P. syringae. Deletion of the CYP107AK1 gene abolished production of coronafacoyl-isoleucine (CFA-Ile), the primary coronafacoyl phytotoxin produced by S. scabiei. Structural elucidation of accumulated biosynthetic intermediates in the ΔCYP107AK1 mutant indicated that CYP107AK1 is required for introducing the oxygen atom that ultimately forms the carbonyl group in the CFA backbone. The CYP107AK1 gene along with two additional genes involved in CFA-Ile biosynthesis in S. scabiei were found to be associated with putative CFA biosynthetic genes in other actinobacteria but not in other organisms. Analysis of the overall genetic content and organization of known and putative CFA biosynthetic gene clusters, together with phylogenetic analysis of the core biosynthetic genes, indicates that horizontal gene transfer has played an important role in the dissemination of the gene cluster and that rearrangement, insertion, and/or deletion events have likely contributed to the divergent biosynthetic evolution of coronafacoyl phytotoxins in bacteria. IMPORTANCE The ability of plants to defend themselves against invading pathogens relies on complex signaling pathways that are controlled by key phytohormones such as jasmonic acid (JA). Some phytopathogenic bacteria have evolved the ability to manipulate JA signaling in order to overcome host defenses by producing coronatine (COR), which functions as a potent JA mimic. COR and COR-like molecules, collectively referred to as coronafacoyl phytotoxins, are produced by several different plant-pathogenic bacteria, and this study provides supporting evidence that different biosynthetic pathways are utilized by different bacteria for production of these phytotoxins. In addition, our study provides a greater understanding of how coronafacoyl phytotoxin biosynthesis may have evolved in phylogenetically distinct bacteria, and we demonstrate that production of these compounds may be more widespread than previously recognized and that their role for the producing organism may not be limited to host-pathogen interactions.

scholarly journals Characterization of the Coronatine-Like Phytotoxins Produced by the Common Scab Pathogen Streptomyces scabies

2015 ◽  
Vol 28 (4) ◽  
pp. 443-454 ◽  
Author(s):  
Joanna K. Fyans ◽  
Mead S. Altowairish ◽  
Yuting Li ◽  
Dawn R. D. Bignell
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Common Scab ◽  
Biosynthetic Gene Cluster ◽  
Major Product ◽  
Streptomyces Scabies ◽  
Coronafacic Acid ◽  
Thaxtomin A ◽  
Metabolites Production

Streptomyces scabies is an important causative agent of common scab disease of potato tubers and other root crops. The primary virulence factor produced by this pathogen is a phytotoxic secondary metabolite called thaxtomin A, which is essential for disease development. In addition, the genome of S. scabies harbors a virulence-associated biosynthetic gene cluster called the coronafacic acid (CFA)-like gene cluster, which was previously predicted to produce metabolites that resemble the Pseudomonas syringae coronatine (COR) phytotoxin. COR consists of CFA linked to an ethylcyclopropyl amino acid called coronamic acid, which is derived from L-allo-isoleucine. Using a combination of genetic and chemical analyses, we show that the S. scabies CFA-like gene cluster is responsible for producing CFA-L-isoleucine as the major product as well as other minor COR-like metabolites. Production of the metabolites was shown to require the cfl gene, which is located within the CFA-like gene cluster and encodes an enzyme involved in ligating CFA to its amino acid partner. CFA-L-isoleucine purified from S. scabies cultures was shown to exhibit bioactivity similar to that of COR, though it was found to be less toxic than COR. This is the first report demonstrating the production of coronafacoyl phytotoxins by S. scabies, which is the most prevalent scab-causing pathogen in North America.

scholarly journals Identification of the Biosynthetic Gene Cluster for 3-Methylarginine, a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

2010 ◽  
Vol 76 (8) ◽  
pp. 2500-2508 ◽  
Author(s):  
S. D. Braun ◽  
J. Hofmann ◽  
A. Wensing ◽  
M. S. Ullrich ◽  
H. Weingart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biosynthetic Gene Cluster ◽  
Pentanoic Acid ◽  
Promising Candidate ◽  
Biosynthesis Gene Cluster ◽  
E Coli ◽  
Highly Active ◽  
Putative Aminotransferase

ABSTRACT The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km , 7 mM; k cat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

Distinguishment of Weak Interactions of Hydrogen Atoms Bound to Carbon Atoms: X-Ray Crystal Structural and Hirshfeld Surface Analyses of 2-Hydroxy-7-methoxy-3-(2,4,6-trimethylbenzoyl)naphthalene with the 2-Methoxylated Homologue

2021 ◽  
Vol 19 ◽  
Author(s):  
Kikuko Iida ◽  
Toyokazu Muto ◽  
Miyuki Kobayashi ◽  
Hiroaki Iitsuka ◽  
Kun Li ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Title Compound ◽  
Molecular Conformation ◽  
Weak Interactions ◽  
Carbonyl Oxygen ◽  
Hirshfeld Surface ◽  
Molecular Surface ◽  
Hydrogen Atoms ◽  
X Ray ◽  
Classical Hydrogen Bond

Abstract: X-ray crystal and Hirshfeld surface analyses of 2-hydroxy-7-methoxy-3-(2,4,6-trimethylbenzoyl)naphthalene and its 2-methoxylated homologue show quantitatively and visually distinct molecular contacts in crystals and minute differences in the weak intermolecular interactions. The title compound has a helical tubular packing, where molecules are piled in a two-folded head-to-tail fashion. The homologue has a tight zigzag molecular string lined up behind each other via nonclassical intermolecular hydrogen bonds between the carbonyl oxygen atom and the hydrogen atom of the naphthalene ring. The dnorm index obtained from the Hirshfeld surface analysis quantitatively demonstrates stronger molecular contacts in the homologue, an ethereal compound, than in the title compound, an alcohol, which is consistent with the higher melting temperature of the former than the latter. Stabilization through the significantly weak intermolecular nonclassical hydrogen bonding interactions in the homologue surpasses the stability imparted by the intramolecular C=O…H–O classical hydrogen bonds in the title compound. The classical hydrogen bond places the six-membered ring in the concave of the title molecule. The hydroxy group opposingly disturbs the molecular aggregation of the title compound, as demonstrated by the distorted H…H interactions covering the molecular surface, owing to the rigid molecular conformation. The position of effective interactions predominate over the strength of the classical/nonclassical hydrogen bonds in the two compounds.

Analysis of Amide Bond Formation with an α-Hydroxy-β-amino Acid Derivative, 3-Amino-2-hydroxy-4-phenylbutanoic Acid, as an Acyl Component:  Byproduction of Homobislactone

2001 ◽  
Vol 66 (16) ◽  
pp. 5537-5544 ◽  
Author(s):  
Yoshio Hayashi ◽  
Yuko Kinoshita ◽  
Koushi Hidaka ◽  
Aiko Kiso ◽  
Hirokazu Uchibori ◽  
...  
Keyword(s):  
Amino Acid ◽  
Acid Derivative ◽  
Bond Formation ◽  
Amide Bond ◽  
Amino Acid Derivative ◽  
Amide Bond Formation

Oxidative cyclization for the synthesis of complex tetrahydrofuran-containing natural products

2013 ◽  
Vol 85 (6) ◽  
pp. 1175-1184 ◽  
Author(s):  
Robert D. C. Pullin ◽  
Radosław M. Lipiński ◽  
Timothy J. Donohoe
Keyword(s):  
Natural Products ◽  
Amino Acid ◽  
Functional Groups ◽  
Lewis Acid ◽  
Excitatory Amino Acid ◽  
Oxidative Cyclization ◽  
Annonaceous Acetogenins ◽  
Hydride Shift ◽  
Vicinal Diols ◽  
Shift Sequence

The osmium-catalyzed oxidative cyclization of vicinal diols onto proximal olefins to generate 2,5-cis-substituted tetrahydrofurans (THFs) has been exploited as the key step for the construction of several complex THF-containing natural products, namely, the annonaceous acetogenins cis-sylvaticin, sylvaticin, and the excitatory amino acid neo-dysiherbaine A. Recently modified conditions that employ a Lewis acid enable the cyclization to proceed under milder conditions, providing greater tolerance to acid-sensitive functional groups, as demonstrated in two of the syntheses. Flexibility for the construction of 2,5-trans-THFs was demonstrated in the synthesis of sylvaticin by utilization of an intramolecular hydride-shift sequence.

scholarly journals New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine

1983 ◽  
Vol 215 (2) ◽  
pp. 253-260 ◽  
Author(s):  
S P Leytus ◽  
W L Patterson ◽  
W F Mangel
Keyword(s):  
Amino Acid ◽  
Kinetic Constants ◽  
Amide Bond ◽  
Single Amino Acid ◽  
Amino Acid Derivative ◽  
Specific Substrate ◽  
Serine Proteinases ◽  
Bovine Trypsin ◽  
Human Thrombin ◽  
Derivatives Of

A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of a single amide bond converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Macroscopic kinetic constants for the interaction of these substrates with bovine trypsin, human and dog plasmin, and human thrombin are reported. Certain of these substrates exhibit extremely large specificity constants. For example, the kcat./Km for bovine trypsin with bis-(N-benzyloxycarbonylglycyl-argininamido)-Rhodamine [(Cbz-Gly-Arg-NH)2-Rhodamine] is 1 670 000 M-1 X S-1. Certain of these substrates are also highly selective. For example, the most specific substrate for human plasmin, (Cbz-Phe-Arg-NH2)-Rhodamine, is not hydrolysed by human thrombin, and the most specific substrate for human thrombin, (Cbz-Pro-Arg-NH)2-Rhodamine, is one of the least specific substrates for human plasmin. Comparison of the kinetic constants for hydrolysis of the dipeptide substrates with that of the single amino acid derivative, (Cbz-Arg-NH)2-Rhodamine, indicates that selection of the proper amino acid residue in the P2 position can effect large increases in substrate specificity. This occurs primarily as a result of an increase in kcat. as opposed to a decrease in Km and, in certain cases, is accompanied by a large increase in selectivity. Because of their high degree of sensitivity and selectivity, these Rhodamine-based dipeptide compounds should be extremely useful substrates for studying serine proteinases.

Reactivity of amino acid anions with nitrogen and oxygen atoms

2018 ◽  
Vol 20 (7) ◽  
pp. 4990-4996 ◽  
Author(s):  
Zhe-Chen Wang ◽  
Ya-Ke Li ◽  
Sheng-Gui He ◽  
Veronica M. Bierbaum
Keyword(s):  
Amino Acid ◽  
Gas Phase ◽  
Ground State ◽  
Phase Reaction ◽  
Gas Phase Reaction ◽  
Oxygen Atoms

Gas-phase reaction of deprotonated tyrosine with a ground state O atom generates five ionic products.

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