scholarly journals The sypA, sypB, and sypC Synthetase Genes Encode Twenty-Two Modules Involved in the Nonribosomal Peptide Synthesis of Syringopeptin by Pseudomonas syringae pv. syringae B301D

2003 ◽  
Vol 16 (4) ◽  
pp. 271-280 ◽  
Author(s):  
Brenda K. Scholz-Schroeder ◽  
Jonathan D. Soule ◽  
Dennis C. Gross
Keyword(s):  
Amino Acids ◽  
Pseudomonas Syringae ◽  
Open Reading Frames ◽  
Acid Activation ◽  
Linear Type ◽  
Nonribosomal Peptide ◽  
Peptide Cyclization ◽  
Peptide Synthetases ◽  
Binding Pockets ◽  
Plant Pathogen Interaction

Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group. Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring. The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.

scholarly journals A Nonribosomal Peptide Synthetase Gene (mgoA) of Pseudomonas syringae pv. syringae Is Involved in Mangotoxin Biosynthesis and Is Required for Full Virulence

2007 ◽  
Vol 20 (5) ◽  
pp. 500-509 ◽  
Author(s):  
Eva Arrebola ◽  
Francisco M. Cazorla ◽  
Diego Romero ◽  
Alejandro Pérez-García ◽  
Antonio de Vicente
Keyword(s):  
Pseudomonas Syringae ◽  
Chromosomal Region ◽  
Open Reading Frames ◽  
Modular Architecture ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Large Gene ◽  
Peptide Synthetase ◽  
Apical Necrosis ◽  
Reading Frames

Pseudomonas syringae pv. syringae, which causes the bacterial apical necrosis of mango, produces the antimetabolite mangotoxin. We report here the cloning, sequencing, and identity analysis of a chromosomal region of 11.1 kb from strain P. syringae pv. syringae UMAF0158, which is involved in mangotoxin biosynthesis. This chromosomal region contains six complete open reading frames (ORFs), including a large gene (ORF5) with a modular architecture characteristic of nonribosomal peptide synthetases (NRPS) named mgoA. A Tn 5 mutant disrupted in mgoA was defective in mangotoxin production, revealing the involvement of the putative NRPS gene in the biosynthesis of mangotoxin. This derivative strain impaired in mangotoxin production also showed a reduction in virulence as measured by necrotic symptoms on tomato leaflets. Mangotoxin production and virulence were restored fully in the NRPS mutant by complementation with plasmid pCG2-6, which contains an 11,103-bp chromosomal region cloned from the wildtype strain P. syringae pv. syringae UMAF0158 that includes the putative NPRS gene (mgoA). The results demonstrate that mgoA has a role in the virulence of P. syringae pv. syringae. The involvement of an NRPS in the production of an antimetabolite toxin from P. syringae inhibiting ornithine acetyltransferase activity is proposed.

scholarly journals The Contribution of Syringopeptin and Syringomycin to Virulence of Pseudomonas syringae pv. syringae strain B301D on the Basis of sypA and syrB1 Biosynthesis Mutant Analysis

2001 ◽  
Vol 14 (3) ◽  
pp. 336-348 ◽  
Author(s):  
Brenda K. Scholz-Schroeder ◽  
Michael L. Hutchison ◽  
Ingeborg Grgurina ◽  
Dennis C. Gross
Keyword(s):  
Pseudomonas Syringae ◽  
High Performance ◽  
Sweet Cherry ◽  
Peptide Structure ◽  
Acid Activation ◽  
Virulence Determinants ◽  
Amino Acid Activation ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Peptide Biosynthesis

Sequencing of an approximately 3.9-kb fragment downstream of the syrD gene of Pseudomonas syringae pv. syringae strain B301D revealed that this region, designated sypA, codes for a peptide synthetase, a multifunctional enzyme involved in the thiotemplate mechanism of peptide biosynthesis. The translated protein sequence encompasses a complete amino acid activation module containing the conserved domains characteristic of peptide synthetases. Analysis of the substrate specificity region of this module indicates that it incorporates 2,3-dehydroaminobutyric acid into the syringopeptin peptide structure. Bioassay and high performance liquid chromatography data confirmed that disruption of the sypA gene in strain B301D resulted in the loss of syringopeptin production. The contribution of syringopeptin and syringomycin to the virulence of P. syringae pv. syringae strain B301D was examined in immature sweet cherry with sypA and syrB1 synthetase mutants defective in the production of the two toxins, respectively. Syringopeptin (sypA) and syringomycin (syrB1) mutants were reduced in virulence 59 and 26%, respectively, compared with the parental strain in cherry, whereas the syringopeptin-syringomycin double mutant was reduced 76% in virulence. These data demonstrate that syringopeptin and syringomycin are major virulence determinants of P. syringae pv. syringae.

scholarly journals Bacterial-like nonribosomal peptide synthetases produce cyclopeptides in the zygomycetous fungus Mortierella alpina

2020 ◽  
Author(s):  
Jacob M. Wurlitzer ◽  
Aleksa Stanišić ◽  
Ina Wasmuth ◽  
Sandra Jungmann ◽  
Dagmar Fischer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Natural Products ◽  
Mortierella Alpina ◽  
Bioactive Molecules ◽  
Fungal Genome ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases ◽  
Bacterial Origin ◽  
Basal Fungi

AbstractFungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such as Mortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfey’s method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. This is the first report of nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products.IMPORTANCEFungal natural compounds are industrially produced with application in antibiotic treatment, cancer medications and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but re-identification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, denoted malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much closer related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. Both enzymes are the first biochemically characterized natural product biosynthesis enzymes of basal fungi. Hence, this report establishes early diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

scholarly journals Cloning, Sequencing, and Characterization of the Iturin A Operon

2001 ◽  
Vol 183 (21) ◽  
pp. 6265-6273 ◽  
Author(s):  
Kenji Tsuge ◽  
Takanori Akiyama ◽  
Makoto Shoda
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fatty Acid Synthetase ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Upstream Sequence ◽  
Flanking Sequence ◽  
Coding Region ◽  
Iturin A ◽  
Peptide Synthetases

ABSTRACT Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence ared-Ser→l-Asn, while in iturin A these amino acids are inverted (i.e., d-Asn→l-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, andituB have high levels of homology to the counterpart genesfenF (79%), mycA (79%), and mycB(79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC andmycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.

Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino Acids

2014 ◽  
Vol 126 (38) ◽  
pp. 10269-10272 ◽  
Author(s):  
Hajo Kries ◽  
Rudolf Wachtel ◽  
Anja Pabst ◽  
Benedikt Wanner ◽  
David Niquille ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

Generality of Peptide Cyclization Catalyzed by Isolated Thioesterase Domains of Nonribosomal Peptide Synthetases†

Biochemistry ◽  
2001 ◽  
Vol 40 (24) ◽  
pp. 7099-7108 ◽  
Author(s):  
Rahul M. Kohli ◽  
John W. Trauger ◽  
Dirk Schwarzer ◽  
Mohamed A. Marahiel ◽  
Christopher T. Walsh
Keyword(s):  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Cyclization ◽  
Peptide Synthetases

Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino Acids

2014 ◽  
Vol 53 (38) ◽  
pp. 10105-10108 ◽  
Author(s):  
Hajo Kries ◽  
Rudolf Wachtel ◽  
Anja Pabst ◽  
Benedikt Wanner ◽  
David Niquille ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

scholarly journals Bacterial-like nonribosomal peptide synthetases produce cyclopeptides in the zygomycetous fungus Mortierella alpina

2020 ◽  
Author(s):  
Jacob M. Wurlitzer ◽  
Aleksa Stanišić ◽  
Ina Wasmuth ◽  
Sandra Jungmann ◽  
Dagmar Fischer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Natural Products ◽  
Mortierella Alpina ◽  
Bioactive Molecules ◽  
Fungal Genome ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases ◽  
Bacterial Origin ◽  
Basal Fungi

Fungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such as Mortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfey's method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as yet unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products. IMPORTANCE Fungal natural compounds are industrially produced with application in antibiotic treatment, cancer medications and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but re-identification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, denoted malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much closer related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. Both enzymes were biochemically characterized and are involved in as yet unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

Reprogramming Nonribosomal Peptide Synthesis by Surgical Mutation

Synlett ◽  
2019 ◽  
Vol 30 (19) ◽  
pp. 2123-2130
Author(s):  
David L. Niquille ◽  
Douglas A. Hansen ◽  
Donald Hilvert
Keyword(s):  
Amino Acids ◽  
Peptide Synthesis ◽  
High Throughput ◽  
Building Blocks ◽  
Downstream Processing ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Nonribosomal Peptide Synthesis ◽  
A Domains ◽  
High Throughput Assay

Nonribosomal peptide synthetases produce highly modified bioactive peptides, many of which are used therapeutically. As such, they have been the target of intense protein engineering to enable biosynthetic access to peptide variants with improved drug properties or altered bioactivities. In this account, we describe our ongoing efforts to reprogram nonribosomal peptide synthesis by surgical mutation. In contrast to ribosomal biosynthesis, nonribosomal peptide synthesis has proven difficult to engineer, arguably due to a lack of suitable tools. To address this limitation, we have established a high-throughput assay that provides unprecedented control over the gatekeeper adenylation domains responsible for building block selection and incorporation. Expansion of this strategy to other building blocks and domains promises to make it a powerful evolutionary platform for tailoring assembly lines for custom synthesis of peptide therapeutics.1. Nonribosomal Peptides2. Reprogramming A Domains for Clickable Amino Acids3 A High-Throughput A Domain Assay4 Reprogramming A Domains for β-Amino Acids5 Downstream Processing6 Conclusions and Outlook

scholarly journals Substrate Specificity of the Nonribosomal Peptide Synthetase PvdD from Pseudomonas aeruginosa

2003 ◽  
Vol 185 (9) ◽  
pp. 2848-2855 ◽  
Author(s):  
David F. Ackerley ◽  
Tom T. Caradoc-Davies ◽  
Iain L. Lamont
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Three Dimensional ◽  
Protein Product ◽  
Synthetase Activity ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Three Dimensional Modeling ◽  
Peptide Synthetase

ABSTRACT Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdinePAO, which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified. l-Threonine, one of the amino acid residues in pyoverdinePAO, was an effective substrate for the recombinant protein in ATP-PPi exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including d-threonine, l-serine, and l-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases.

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