Dataset Information

Structure of a eukaryotic nonribosomal peptide synthetase adenylation domain that activates a large hydroxamate amino acid in siderophore biosynthesis.

ABSTRACT: Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.

SUBMITTER: Lee TV

PROVIDER: S-EPMC2807299 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structure of a eukaryotic nonribosomal peptide synthetase adenylation domain that activates a large hydroxamate amino acid in siderophore biosynthesis.

Lee T Verne TV Johnson Linda J LJ Johnson Richard D RD Koulman Albert A Lane Geoffrey A GA Lott J Shaun JS Arcus Vickery L VL

The Journal of biological chemistry 20091118 4

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L- ...[more]

PMID: 19923209

Similar Datasets

Project description:Recent papers have reported dipeptides containing d-amino acids to have novel effects that cannot be observed with ll-dipeptides, and such dipeptides are expected to be novel functional compounds for pharmaceuticals and food additives. Although the functions of d-amino acid-containing dipeptides are gaining more attention, there are few reports on the synthetic enzymes that can accept d-amino acids as substrates, and synthetic methods for d-amino acid-containing dipeptides have not yet been constructed. Previously, we developed a chemoenzymatic system for amide synthesis that comprised enzymatic activation and a subsequent nucleophilic substitution reaction. In this study, we demonstrated the application of the system for d-amino acid-containing-dipeptide synthesis. We chose six adenylation domains as targets according to our newly constructed hypothesis, i.e., an adenylation domain located upstream from the epimerization domain may activate d-amino acid as well as l-amino acid. We successfully synthesized over 40 kinds of d-amino acid-containing dipeptides, including ld-, dl-, and dd-dipeptides, using only two adenylation domains, TycA-A from tyrocidine synthetase and BacB2-A from bacitracin synthetase. Furthermore, this study offered the possibility that the epimerization domain could be a clue to the activity of the adenylation domains toward d-amino acid. This paper provides additional information regarding d-amino acid-containing-dipeptide synthesis through the combination of enzymatic adenylation and chemical nucleophilic reaction, and this system will be a useful tool for dipeptide synthesis.IMPORTANCE Because almost all amino acids in nature are l-amino acids, the functioning of d-amino acids has received little attention. Thus, there is little information available on the activity of enzymes toward d-amino acids or synthetic methods for d-amino acid-containing dipeptides. Recently, d-amino acids and d-amino acid-containing peptides have attracted attention as novel functional compounds, and d-amino acid-activating enzymes and synthetic methods are required for the development of the d-amino acid-containing-peptide industry. This study provides additional knowledge regarding d-amino acid-activating enzymes and proposes a unique synthetic method for d-amino acid-containing peptides, including ld-, dl-, and dd-dipeptides.

Project description: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. N5-acetyl-N5-hydroxy-l-ornithine (l-AHO) and N5-cis-anhydromevalonyl-N5 -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.

Project description:Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single-module nonribosomal peptide synthetases (NRPSs) have been identified to be involved in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking, and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives.One-sentence summaryThe enzymatic basis and mechanistic insights into AneB, a single-module NRPS, highlight its capacity to generate various terpene-amino acid meroterpenoid derivatives.

Project description:Albicidin is a pathotoxin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald disease of sugarcane. Albicidin is synthesized by a nonribosomal pathway via modular polyketide synthase and nonribosomal peptide synthetase (NRPS) megasynthases, and NRPS adenylation (A) domains are responsible for the recognition and activation of specific amino acid substrates. DNA fragments (0.5 kb) encoding the regions responsible for the substrate specificities of six albicidin NRPS A domains from 16 strains of X. albilineans representing the known diversity of this pathogen were amplified and sequenced. Polymorphism analysis of these DNA fragments at different levels (DNA, protein, and NRPS signature) showed that these pathogenicity loci were highly conserved. The conservation of these loci most likely reflects purifying selective pressure, as revealed by a comparison with the variability of nucleotide and amino acid sequences of two housekeeping genes (atpD and efp) of X. albilineans. Nevertheless, the 16 strains of X. albilineans were differentiated into several groups by a phylogenetic analysis of the nucleotide sequences corresponding to the NRPS A domains. One of these groups was representative of the genetic diversity previously found within the pathogen by random fragment length polymorphism and amplified fragment length polymorphism analyses. This group, which differed by three single synonymous nucleotide mutations, contained only four strains of X. albilineans that were all involved in outbreaks of sugarcane leaf scald. The amount of albicidin produced in vitro in agar and liquid media varied among the 16 strains of X. albilineans. However, no relationship among the amount of albicidin produced in vitro and the pathotypes and genetic diversity of the pathogen was found. The NRPS loci contributing to the synthesis of the primary structure of albicidin apparently are not involved in the observed pathogenicity differences among strains of X. albilineans.

Dataset Information

Structure of a eukaryotic nonribosomal peptide synthetase adenylation domain that activates a large hydroxamate amino acid in siderophore biosynthesis.

Publications

Structure of a eukaryotic nonribosomal peptide synthetase adenylation domain that activates a large hydroxamate amino acid in siderophore biosynthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets