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.

Outlining the Hidden Curriculum: Perspectives on Successfully Navigating Scientific Conferences.

Scientific conferences and meetings are valuable opportunities for researchers to network, communicate, and develop knowledge. For early career scientists, conferences can also be intimidating, confusing, and overwhelming, especially without having adequate preparation or experience. In this Perspective, we provide advice based on previous experiences navigating scientific meetings and conferences. These guidelines outline parts of the hidden curriculum around preparing for and attending meetings, navigating conference sessions, networking with other scientists, and participating in social activities while upholding a recommended code of conduct.

Expanding the Utility of Bioinformatic Data for the Full Stereostructural Assignments of Marinolides A and B, 24- and 26-Membered Macrolactones Produced by a Chemically Exceptional Marine-Derived Bacterium.

Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures initially assigned by bioinformatic data analysis. Macrolactones typically possess complex stereochemistry, the assignments of which have been one of the most difficult undertakings in natural products chemistry, and in most cases, the use of X-ray diffraction methods and total synthesis have been the major methods of assigning their absolute configurations. More recently, however, it has become apparent that the integration of bioinformatic data is growing in utility to assign absolute configurations. Genome mining and bioinformatic analysis identified the 97 kb mld biosynthetic cluster harboring seven type I polyketide synthases. A detailed bioinformatic investigation of the ketoreductase and enoylreductase domains within the multimodular polyketide synthases, coupled with NMR and X-ray diffraction data, allowed for the absolute configurations of marinolides A and B to be determined. While using bioinformatics to assign the relative and absolute configurations of natural products has high potential, this method must be coupled with full NMR-based analysis to both confirm bioinformatic assignments as well as any additional modifications that occur during biosynthesis.

Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis.

Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites.

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

Marinoterpins A-C: Rare Linear Merosesterterpenoids from Marine-Derived Actinomycete Bacteria of the Family Streptomycetaceae.

The chemical examination of two undescribed marine actinobacteria has yielded three rare merosesterterpenoids, marinoterpins A-C (1-3, respectively). These compounds were isolated from the culture broth extracts of two marine-derived actinomycetes associated with the family Streptomycetaceae, (our strains were CNQ-253 and AJS-327). The structures of the new compounds were determined by extensive interpretation of 1D and 2D NMR, MS, and combined spectroscopic data. These compounds represent new chemical motifs, combining quinoline-N-oxides with a linear sesterterpenoid side chain. Additionally, consistent in all three metabolites is the rare occurrence of two five-ring ethers, which were derived from an apparent cyclization of methyl group carbons to adjacent hydroxy-bearing methylene groups in the sesterterpenoid side chain. Genome scanning of AJS-327 allowed for the identification of the marinoterpin (mrt) biosynthetic cluster, which consists of 16 open-reading frames that code for a sesterterpene pyrophosphate synthase, prenyltransferase, type II polyketide synthase, anthranilate:CoA-ligase, and several tailoring enzymes apparently responsible for installing the N-oxide and bis-tetrahydrofuran ring motifs.

Azetidine-Bearing Non-Ribosomal Peptides, Bonnevillamides D and E, Isolated from a Carrion Beetle-Associated Actinomycete.

Two new nonribosomal peptides, bonnevillamides D and E (1 and 2), have been discovered in Streptomyces sp. UTZ13 isolated from the carrion beetle, Nicrophorus concolor. Combinational analysis of the UV, MS, and NMR spectroscopic data revealed that their planar structures were comprised of dichlorinated linear peptides containing nonproteinogenic amino acid residues, such as 4-methylazetidinecarboxylic acid and 4-O-acetyl-5-methylproline. The configurations of bonnevillamides D and E (1 and 2) were determined based on ROESY correlations, the advanced Marfey's method, phenylglycine methyl ester derivatization, molecular modeling, and circular dichroism spectroscopy. The nonribosomal peptide synthetase biosynthetic pathway of bonnevillamides D and E has been proposed using bioinformatic analysis of the whole-genome sequence data of Streptomyces sp. UTZ13. Their biological activity toward the aggregation of amyloid-ß, which is one of the key pathogenic proteins in Alzheimer's disease, was evaluated using a thioflavin T assay and gel electrophoresis. Bonnevillamides D and E reversed the fibril formation by inducing the monomerization of amyloid-ß aggregates.

Emerimicins V-X, 15-Residue Peptaibols Discovered from an Acremonium sp. through Integrated Genomic and Chemical Approaches.

Fermentation of Acremonium tubakii W. Gams isolated from a soil sample collected from the University of Utah led to the isolation and characterization of six new linear pentadecapeptides, emerimicins V-X (1-6). Peptaibols containing 15-residues are quite rare, with only 22 reported. Genome mining and bioinformatic analysis were used to identify the emerimicin 60 kbp eme biosynthetic cluster harboring a single 16-module hybrid polyketide-nonribosomal peptide synthetase. A detailed bioinformatic investigation of the corresponding 15 adenylation domains, combined with 1D and 2D NMR experiments, LC-MS/MS data, and advanced Marfey's method, allowed for the elucidation and absolute configuration of all proteinogenic and nonproteinogenic amino acid residues in 1-6. As some peptaibols possess cytotoxic activity, a zebrafish embryotoxicity assay was used to evaluate the toxicity of the six emerimicins and showed that emerimicin V (1) and VI (2) exhibit the most potent activity. Additionally, out of the six emerimicins, 1 displayed modest activity against Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with MIC values of 64, 32, and 64 µg/mL, respectively.

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse.

Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature's halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases.

Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precisely positioned olefin from the unnatural amino acid is proposed to facilitate a scaffold rearrangement of the PKS-NRPS product to forge the maleimide and succinimide cores of oxaleimides.

Bonnevillamides, Linear Heptapeptides Isolated from a Great Salt Lake-Derived Streptomyces sp.

Streptomyces sp. GSL-6B was isolated from sediment collected from the Great Salt Lake and investigation of its organic extract led to the isolation of three new linear heptapeptides, bonnevillamides A (1), B (2), and C (3). The bonnevillamides represent a new class of linear peptides featuring unprecedented non-proteinogenic amino acids. All three peptides contain the newly characterized bonnevillic acid moiety (3-(3,5-dichloro-4-methoxyphenyl)-2-hydroxyacrylic acid), as well as a heavily modified proline residue. Moreover, in bonnevillamide A, the terminal proline residue found in bonnevillamides B and C is replaced with 4-methyl-azetidine-2-carboxylic acid methyl ester. The structures of the three heptapeptides were elucidated by NMR, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and LC-MS/MS, and the absolute configuration of all proteinogenic amino acid residues were determined by advanced Marfey's method. Bonnevillamides A, B and C were evaluated for their effects on zebrafish embryo development. All three heptapeptides were shown to modulate heart growth and cardiac function, with bonnevillamide B having the most pronounced effect.

Biochemical and Structural Basis for Controlling Chemical Modularity in Fungal Polyketide Biosynthesis.

Modular collaboration between iterative fungal polyketide synthases (IPKSs) is an important mechanism for generating structural diversity of polyketide natural products. Inter-PKS communication and substrate channeling are controlled in large by the starter unit acyl carrier protein transacylase (SAT) domain found in the accepting IPKS module. Here, we reconstituted the modular biosynthesis of the benzaldehyde core of the chaetoviridin and chaetomugilin azaphilone natural products using the IPKSs CazF and CazM. Our studies revealed a critical role of CazM's SAT domain in selectively transferring a highly reduced triketide product from CazF. In contrast, a more oxidized triketide that is also produced by CazF and required in later stages of biosynthesis of the final product is not recognized by the SAT domain. The structural basis for the acyl unit selectivity was uncovered by the first X-ray structure of a fungal SAT domain, highlighted by a covalent hexanoyl thioester intermediate in the SAT active site. The crystal structure of SAT domain will enable protein engineering efforts aimed at mixing and matching different IPKS modules for the biosynthesis of new compounds.

Comparison of 10,11-Dehydrocurvularin Polyketide Synthases from Alternaria cinerariae and Aspergillus terreus Highlights Key Structural Motifs.

Iterative type I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A phytotoxic macrolide with anticancer properties, 10,11-dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non-reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast, and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis and homology modeling of these enzymes identified variable surfaces and conserved motifs that are implicated in product formation.

Resistance Gene Association and Inference Network (ReGAIN): A Bioinformatics Pipeline for Assessing Probabilistic Co-Occurrence Between Resistance Genes in Bacterial Pathogens.

The rampant rise of multidrug resistant (MDR) bacterial pathogens poses a severe health threat, necessitating innovative tools to unravel the complex genetic underpinnings of antimicrobial resistance. Despite significant strides in developing genomic tools for detecting resistance genes, a gap remains in analyzing organism-specific patterns of resistance gene co-occurrence. Addressing this deficiency, we developed the Resistance Gene Association and Inference Network (ReGAIN), a novel web-based and command line genomic platform that uses Bayesian network structure learning to identify and map resistance gene networks in bacterial pathogens. ReGAIN not only detects resistance genes using well-established methods, but also elucidates their complex interplay, critical for understanding MDR phenotypes. Focusing on ESKAPE pathogens, ReGAIN yielded a queryable database for investigating resistance gene co-occurrence, enriching resistome analyses, and providing new insights into the dynamics of antimicrobial resistance. Furthermore, the versatility of ReGAIN extends beyond antibiotic resistance genes to include assessment of co-occurrence patterns among heavy metal resistance and virulence determinants, providing a comprehensive overview of key gene relationships impacting both disease progression and treatment outcomes.

Bacterial diversity and chemical ecology of natural product-producing bacteria from Great Salt Lake sediment.

Great Salt Lake (GSL), located northwest of Salt Lake City, UT, is the largest terminal lake in the USA. While the average salinity of seawater is ~3.3%, the salinity in GSL ranges between 5% and 28%. In addition to being a hypersaline environment, GSL also contains toxic concentrations of heavy metals, such as arsenic, mercury, and lead. The extreme environment of GSL makes it an intriguing subject of study, both for its unique microbiome and its potential to harbor novel natural product-producing bacteria, which could be used as resources for the discovery of biologically active compounds. Though work has been done to survey and catalog bacteria found in GSL, the Lake's microbiome is largely unexplored, and little to no work has been done to characterize the natural product potential of GSL microbes. Here, we investigate the bacterial diversity of two important regions within GSL, describe the first genomic characterization of Actinomycetota isolated from GSL sediment, including the identification of two new Actinomycetota species, and provide the first survey of the natural product potential of GSL bacteria.

The Discovery of Acremochlorins O-R from an Acremonium sp. through Integrated Genomic and Molecular Networking.

The fermentation of a soil-derived fungus Acremonium sp. led to the isolation of thirteen ascochlorin congeners through integrated genomic and Global Natural Product Social (GNPS) molecular networking. Among the isolated compounds, we identified two unusual bicyclic types, acremochlorins O (1) and P (2), as well as two linear types, acremochlorin Q (3) and R (4). Compounds 1 and 2 contain an unusual benzopyran moiety and are diastereoisomers of each other, the first reported for the ascochlorins. Additionally, we elucidated the structure of 5, a 4-chloro-5-methylbenzene-1,3-diol with a linear farnesyl side chain, and confirmed the presence of eight known ascochlorin analogs (6-13). The structures were determined by the detailed interpretation of 1D and 2D NMR spectroscopy, MS, and ECD calculations. Compounds 3 and 9 showed potent antibacterial activity against Staphylococcus aureus and Bacillus cereus, with MIC values ranging from 2 to 16 µg/mL.

Conformational Control in Diterpene Synthase TlnA: Elucidating the Mechanism of 5-8-6 Skeleton Formation through Ring Expansion.

TlnA produces a distinct cyclohexane-fused 5-8-6 ring system, different from the prevalent 5-8-5 scaffold synthesized by well-established enzymes. This study identifies two conformations of a carbocation intermediate, revealing how the enzyme environment prohibits one conformation due to steric hindrance, thereby directing the formation of the 5-8-6 system over the 5-8-5 scaffold. This investigation enhances our understanding of diterpene biosynthesis and the impact of enzyme environments on chemical reactions, providing valuable insights into the formation of complex cyclic structures.

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.

Bacterial Diversity and Chemical Ecology of Natural Product-Producing Bacteria from Great Salt Lake Sediment.

Great Salt Lake (GSL), located northwest of Salt Lake City, UT, is the largest terminal lake in the United States. While the average salinity of seawater is ~3.3%, the salinity in GSL ranges between 5-28%. In addition to being a hypersaline environment, GSL also contains toxic concentrations of heavy metals, such as arsenic, mercury, and lead. The extreme environment of GSL makes it an intriguing subject of study, both for its unique microbiome and its potential to harbor novel natural product-producing bacteria, which could be used as resources for the discovery of biologically active compounds. Though work has been done to survey and catalogue bacteria found in GSL, the Lake's microbiome is largely unexplored, and little-to-no work has been done to characterize the natural product potential of GSL microbes. Here, we investigate the bacterial diversity of two important regions within GSL, describe the first genomic characterization of Actinomycetota isolated from GSL sediment, including the identification of a new Saccharomonospora species, and provide the first survey of the natural product potential of GSL bacteria.

Identification and characterization of the chaetoviridin and chaetomugilin gene cluster in Chaetomium globosum reveal dual functions of an iterative highly-reducing polyketide synthase.

We report the identification and characterization of the caz biosynthetic cluster from Chaetomium globosum and the characterization of a highly reducing polyketide synthase (PKS) that acts in both a sequential and convergent manner with a nonreducing PKS to form the chaetomugilin and chaetoviridin azaphilones. Genetic inactivation studies verified the involvement of individual caz genes in the biosynthesis of the azaphilones. Through in vitro reconstitution, we demonstrated the in vitro synthesis of chaetoviridin A from the pyranoquinone intermediate cazisochromene using the highly reducing PKS and an acyltransferase.