Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains.

Covering: 1997 to July 2023The adenylation reaction has been a subject of scientific intrigue since it was first recognized as essential to many biological processes, including the homeostasis and pathogenicity of some bacteria and the activation of amino acids for protein synthesis in mammals. Several foundational studies on adenylation (A) domains have facilitated an improved understanding of their molecular structures and biochemical properties, in particular work on nonribosomal peptide synthetases (NRPSs). In NRPS pathways, A domains activate their respective acyl substrates for incorporation into a growing peptidyl chain, and many nonribosomal peptides are bioactive. From a natural product drug discovery perspective, improving existing bioinformatics platforms to predict unique NRPS products more accurately from genomic data is desirable. Here, we summarize characterization efforts of A domains primarily from NRPS pathways from July 1997 up to July 2023, covering protein structure elucidation, in vitro assay development, and in silico tools for improved predictions.

Biosynthesis of the fungal nonribosomal peptide penilumamide A and biochemical characterization of a pterin-specific adenylation domain.

We report the characterization of the penilumamide biosynthetic cluster from Aspergillus flavipes CNL-338. In vitro reconstitution experiments demonstrated that three nonribosomal peptide synthetases are required for constructing the tripeptide and studies with dissected adenylation domains allowed for the first biochemical characterization of a domain that selects a pterin-derived building block.

Identification and Characterization of a Cryptic Bifunctional Type I Diterpene Synthase Involved in Talaronoid Biosynthesis from a Marine-Derived Fungus.

We report the identification of the tnd biosynthetic cluster from the marine-derived fungus Aspergillus flavipes and the in vivo characterization of a cryptic type I diterpene synthase. The heterologous expression of the bifunctional terpene synthase led to the discovery of a diterpene backbone, talarodiene, harboring a benzo[a]cyclopenta[d]cyclooctane tricyclic fused ring system. The conversion of geranylgeranyl diphosphate to talarodiene was investigated using 13C-labeling studies, and stable isotope tracer experiments showed the biotransformation of talarodiene into talaronoid C.

Antifungal natural products.

Natural products are specialized small molecules produced in Nature and play pivotal roles in many cellular processes. These compounds possess exquisite chemical diversity and represent some of the most important pharmaceutical agents in human health care. With the rampant rise of fungal pathogens that are becoming resistant to nearly all clinically available antibiotics, there is an increased urgency to find new antifungal therapies with novel modes of action. To meet this need, we must be able to quickly identify new bioactive chemical scaffolds within complex natural extracts, determine their mechanisms of action, and generate appreciable yields for preclinical studies. In this review, we will highlight naturally derived antifungal agents of clinical importance as well as those with strong potential as leads in drug development.

Discovery and characterization of a cytochalasan biosynthetic cluster from the marine-derived fungus Aspergillus flavipes CNL-338.

Cytochalasans are a large family of well-studied cytotoxic molecules isolated from fungi. Investigation into the organic extract of the marine-derived fungal strain Aspergillus flavipes CNL-338 led to the isolation of seven leucine-containing cytochalasans. Genome mining allowed for the identification of the ffs biosynthetic gene cluster, and genetic inactivation studies verified its involvement in cytochalasan biosynthesis. In addition, comparative analysis of key residues in the binding pocket of core cytochalasan biosynthetic enzymes revealed significant similarities among fungal adenylation domains despite differences in substrate preference. We report the identification of leucine-containing cytochalasans from the marine-derived A. flavipes CNL-338 and the characterization of the ffs biosynthetic cluster as verified by genetic inactivation studies.

Phosphorylation of Tyr-950 in the proteasome scaffolding protein RPN2 modulates its interaction with the ubiquitin receptor RPN13.

Protein substrates are targeted to the 26S proteasome through several ubiquitin receptors. One of these receptors, RPN13, is recruited to the proteasome by binding of its N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain to C-terminal residues of the scaffolding protein RPN2. The RPN13 PRU domain is followed by a flexible linker and a C-terminal deubiquitylase adaptor (DEUBAD) domain, which recruits and activates the deubiquitylase UCH37. Both RPN13 and UCH37 have been implicated in human cancers, and inhibitors of the RPN2-RPN13 interaction are being developed as potential therapeutic anticancer agents. Our current study builds on the recognition that a residue central to the RPN2-RPN13 interaction, RPN2 Tyr-950, is phosphorylated in Jurkat cells. We found that the Tyr-950 phosphorylation enhances binding to RPN13. The crystal structure of the RPN2-RPN13 pTyr-950-ubiquitin complex was determined at 1.76-Å resolution and reveals specific interactions with positively charged side chains in RPN13 that explain how phosphorylation increases binding affinity without inducing conformational change. Mutagenesis and quantitative binding assays were then used to validate the crystallographic interface. Our findings support a model in which RPN13 recruitment to the proteasome is enhanced by phosphorylation of RPN2 Tyr-950, have important implications for efforts to develop specific inhibitors of the RPN2-RPN13 interaction, and suggest the existence of a previously unknown stress-response pathway.