scholarly journals tRNA-Dependent Aminoacylation of an Amino Sugar Intermediate in the Biosynthesis of a Streptothricin-Related Antibiotic

2016 ◽  
Vol 82 (12) ◽  
pp. 3640-3648 ◽  
Author(s):  
Chitose Maruyama ◽  
Haruka Niikura ◽  
Miho Izumikawa ◽  
Junko Hashimoto ◽  
Kazuo Shin-ya ◽  
...  
Keyword(s):  
Structural Diversity ◽  
Amino Sugar ◽  
Peptide Bond ◽  
Nonribosomal Peptide Synthetase ◽  
Side Chain ◽  
Content Type ◽  
Bond Forming ◽  
Peptide Synthetase ◽  
Related Compounds ◽  
Distinct Peptide

ABSTRACTThe antibiotic streptothricin (ST) possesses an amino sugar bound to anl-β-lysine (β-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a β-Lys residue. Here, inStreptomyces luteocolorNBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics.IMPORTANCEThe antibiotic streptothricin (ST) possesses an amino sugar bound to anl-β-lysine (β-Lys) side chain via a peptide bond formed by a nonribosomal peptide synthetase (NRPS). BD-12, an analogue of ST, carries a glycine-derived side chain rather than β-Lys, and here, we describe the BD-12-biosynthetic gene cluster fromStreptomycesluteocolorNBRC13826, which contains theorf11gene encoding a novel tRNA-dependent peptide bond-forming enzyme. The unique Fem-like enzyme (Orf11) accepts the amino sugar as a substrate and mediates the peptide formation between the amino sugar intermediate and glycine. Our studies demonstrate that the structural diversity of the side chains of ST-related compounds in nature is generated via two distinct peptide bond-forming mechanisms.

scholarly journals Structural and Functional Insights into a Peptide Bond-Forming Bidomain from a Nonribosomal Peptide Synthetase

Structure ◽  
2007 ◽  
Vol 15 (7) ◽  
pp. 781-792 ◽  
Author(s):  
Stefan A. Samel ◽  
Georg Schoenafinger ◽  
Thomas A. Knappe ◽  
Mohamed A. Marahiel ◽  
Lars-Oliver Essen
Keyword(s):  
Peptide Bond ◽  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Bond Forming ◽  
Peptide Synthetase

scholarly journals Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosa

2014 ◽  
Vol 80 (18) ◽  
pp. 5723-5731 ◽  
Author(s):  
Mark J. Calcott ◽  
Jeremy G. Owen ◽  
Iain L. Lamont ◽  
David F. Ackerley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Peptide Bond ◽  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Threonine Residue ◽  
Peptide Synthetase ◽  
A Domains ◽  
High Yields ◽  
Acid Substrate

ABSTRACTPyoverdine is a fluorescent nonribosomal peptide siderophore made by fluorescent pseudomonads. ThePseudomonas aeruginosanonribosomal peptide synthetase (NRPS) PvdD contains two modules that each incorporate anl-threonine residue at the C-terminal end of pyoverdine. In an attempt to generate modified pyoverdine peptides, we substituted alternative-substrate-specifying adenylation (A) and peptide bond-catalyzing condensation (C) domains into the second module of PvdD. When just the A domain was substituted, the resulting strains produced only wild-type pyoverdine—at high levels if the introduced A domain specified threonine or at trace levels otherwise. The high levels of pyoverdine synthesis observed whenever the introduced A domain specified threonine indicated that these nonnative A domains were able to communicate effectively with the PvdD C domain. Moreover, the unexpected observation that non-threonine-specifying A domains nevertheless incorporated threonine into pyoverdine suggests that the native PvdD C domain exhibited stronger selectivity than these A domains for the incorporated amino acid substrate (i.e., misactivation of a threonine residue by the introduced A domains was more frequent than misincorporation of a nonthreonine residue by the PvdD C domain). In contrast, substitution of both the C and A domains of PvdD generated high yields of rationally modified pyoverdines in two instances, these pyoverdines having either a lysine or a serine residue in place of the terminal threonine. However, C-A domain substitution more commonly yielded a truncated peptide product, likely due to stalling of synthesis on a nonfunctional recombinant NRPS template.

scholarly journals Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI

2017 ◽  
Vol 199 (21) ◽  
Author(s):  
Lauren M. Petersen ◽  
Kaitlyn LaCourse ◽  
Tim A. Schöner ◽  
Helge Bode ◽  
Louis S. Tisa
Keyword(s):  
Antimicrobial Activity ◽  
Serratia Marcescens ◽  
Virulence Factors ◽  
Nonribosomal Peptide Synthetase ◽  
Mrna Levels ◽  
Insect Pathogen ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Hemolysin Gene ◽  
Peptide Synthetase

ABSTRACT Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens. The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA, these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI. IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences in regulation of common virulence mechanisms between these two species. With the emergence of antibiotic-resistant microorganisms, there is a widespread need to understand the regulation of pathogenesis. The significance of this study is the presentation of evidence for cross-pathway regulation of virulence factors and how the elimination of one mechanism may be compensated for by the upregulation of others.

scholarly journals New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

2012 ◽  
Vol 78 (23) ◽  
pp. 8208-8218 ◽  
Author(s):  
Antonia Gallo ◽  
Kenneth S. Bruno ◽  
Michele Solfrizzo ◽  
Giancarlo Perrone ◽  
Giuseppina Mulè ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Biosynthetic Pathway ◽  
Nonribosomal Peptide Synthetase ◽  
Gene Encoding ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase ◽  
Ochratoxin Α ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACTOchratoxin A (OTA), a mycotoxin produced byAspergillusandPenicilliumspecies, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated inPenicilliumspecies. InAspergillusspecies, onlypksgenes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producingA. carbonariusITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of annrpsgene product in OTA biosynthetic pathway in anAspergillusspecies. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA inA. carbonariusand that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

scholarly journals A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Eileen Brandenburger ◽  
Markus Gressler ◽  
Robin Leonhardt ◽  
Gerald Lackner ◽  
Andreas Habel ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Nonribosomal Peptide Synthetase ◽  
Full Length ◽  
Amide Bond ◽  
White Rot ◽  
Mass Spectrometry Analysis ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase

ABSTRACT The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N 5-acetyl-N 5-hydroxy-l-ornithine (l-AHO) and N 5-cis-anhydromevalonyl-N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography–high-resolution electrospray ionization–mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes. IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.

scholarly journals Draft Genome Sequence of a Porcine Commensal, Rothia nasimurium, Encoding a Nonribosomal Peptide Synthetase Predicted To Produce the Ionophore Antibiotic Valinomycin

2017 ◽  
Vol 5 (22) ◽  
Author(s):  
Rogier A. Gaiser ◽  
Marnix H. Medema ◽  
Michiel Kleerebezem ◽  
Peter van Baarlen ◽  
Jerry M. Wells
Keyword(s):  
Genome Sequence ◽  
Bacillus Pumilus ◽  
Draft Genome ◽  
Nonribosomal Peptide Synthetase ◽  
Draft Genome Sequence ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase

ABSTRACT We report the draft whole-genome sequence of Rothia nasimurium isolated from a porcine tonsil. The genome encodes a nonribosomal peptide synthetase predicted to produce valinomycin, a cyclic dodecadepsipeptide ionophore. Previously, valinomycin was known to be produced only by Streptomyces species and isolates belonging to the Bacillus pumilus group.

scholarly journals Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cornelia Hermes ◽  
René Richarz ◽  
Daniel A. Wirtz ◽  
Julian Patt ◽  
Wiebke Hanke ◽  
...  
Keyword(s):  
Natural Products ◽  
Docking Studies ◽  
Nonribosomal Peptide Synthetase ◽  
Side Chain ◽  
Side Chains ◽  
Protein Inhibitor ◽  
Assembly Lines ◽  
Bioinformatics Analyses ◽  
Peptide Synthetase ◽  
Substrate Promiscuity

AbstractThe potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

scholarly journals Identification of a Gene Involved in the Synthesis of a Dipeptidyl Peptidase IV Inhibitor in Aspergillus oryzae

2012 ◽  
Vol 78 (19) ◽  
pp. 6996-7002 ◽  
Author(s):  
Kazuhiko Imamura ◽  
Yoshihito Tsuyama ◽  
Terukage Hirata ◽  
Sumihiro Shiraishi ◽  
Kazutoshi Sakamoto ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Expression Patterns ◽  
Biosynthetic Gene Cluster ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase ◽  
Isoquinoline Derivative

ABSTRACTWYK-1 is a dipeptidyl peptidase IV inhibitor produced byAspergillus oryzaestrain AO-1. Because WYK-1 is an isoquinoline derivative consisting of threel-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome ofA. oryzaeRIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructedwykNgene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream ofwykNwere similar to the expression pattern ofwykNunder several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose thatwykNand the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.

scholarly journals Cryptic Production oftrans-3-Hydroxyproline in Echinocandin B Biosynthesis

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Johanna Mattay ◽  
Stefanie Houwaart ◽  
Wolfgang Hüttel
Keyword(s):  
Amino Acids ◽  
Nonribosomal Peptide Synthetase ◽  
Peptide Antibiotic ◽  
Prolonged Incubation ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Echinocandin B ◽  
Peptide Synthetase ◽  
Proline Hydroxylation ◽  
Proline Hydroxylase

ABSTRACTEchinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines:trans-4-hydroxy-l-proline and, in most echinocandin structures, (trans-2,3)-3-hydroxy-(trans-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis byGlarea lozoyensis, both amino acids are found in pneumocandin A0, while in pneumocandin B0the latter residue is replaced bytrans-3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis byAspergillusspecies, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster inAspergillus pachycristatus. Surprisingly,l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced byCryptosporiopsis.IMPORTANCESecondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A0and B0are produced by the fungusGlarea lozoyensis. They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A0and B0, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis inAspergillus pachycristatusproduces the same hydroxyprolines, with an increased proportion oftrans-3-hydroxyproline. However, echinocandin B biosynthesis does not requiretrans-3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation inG. lozoyensisandA. pachycristatusprovides an unusual insight into peptide antibiotic biosynthesis—namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.

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