Cytochromes P450 involved in bacterial RiPP biosyntheses.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of secondary metabolites that have garnered scientific attention due to their complex scaffolds with potential roles in medicine, agriculture, and chemical ecology. RiPPs derive from the cleavage of ribosomally synthesized proteins and additional modifications, catalyzed by various enzymes to alter the peptide backbone or side chains. Of these enzymes, cytochromes P450 (P450s) are a superfamily of heme-thiolate proteins involved in many metabolic pathways, including RiPP biosyntheses. In this review, we focus our discussion on P450 involved in RiPP pathways and the unique chemical transformations they mediate. Previous studies have revealed a wealth of P450s distributed across all domains of life. While the number of characterized P450s involved in RiPP biosyntheses is relatively small, they catalyze various enzymatic reactions such as C-C or C-N bond formation. Formation of some RiPPs is catalyzed by more than one P450, enabling structural diversity. With the continuous improvement of the bioinformatic tools for RiPP prediction and advancement in synthetic biology techniques, it is expected that further cytochrome P450-mediated RiPP biosynthetic pathways will be discovered. SUMMARY: The presence of genes encoding P450s in gene clusters for ribosomally synthesized and post-translationally modified peptides expand structural and functional diversity of these secondary metabolites, and here, we review the current state of this knowledge.

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.

Comparative and pangenomic analysis of the genus Streptomyces.

Streptomycetes are highly metabolically gifted bacteria with the abilities to produce bioproducts that have profound economic and societal importance. These bioproducts are produced by metabolic pathways including those for the biosynthesis of secondary metabolites and catabolism of plant biomass constituents. Advancements in genome sequencing technologies have revealed a wealth of untapped metabolic potential from Streptomyces genomes. Here, we report the largest Streptomyces pangenome generated by using 205 complete genomes. Metabolic potentials of the pangenome and individual genomes were analyzed, revealing degrees of conservation of individual metabolic pathways and strains potentially suitable for metabolic engineering. Of them, Streptomyces bingchenggensis was identified as a potent degrader of plant biomass. Polyketide, non-ribosomal peptide, and gamma-butyrolactone biosynthetic enzymes are primarily strain specific while ectoine and some terpene biosynthetic pathways are highly conserved. A large number of transcription factors associated with secondary metabolism are strain-specific while those controlling basic biological processes are highly conserved. Although the majority of genes involved in morphological development are highly conserved, there are strain-specific varieties which may contribute to fine tuning the timing of cellular differentiation. Overall, these results provide insights into the metabolic potential, regulation and physiology of streptomycetes, which will facilitate further exploitation of these important bacteria.

Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses.

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.

Biosynthesis and structures of cyclomarins and cyclomarazines, prenylated cyclic peptides of marine actinobacterial origin.

Two new diketopiperazine dipeptides, cyclomarazines A and B, were isolated and characterized along with the new cyclic heptapeptide cyclomarin D from the marine bacterium Salinispora arenicola CNS-205. These structurally related cyclic peptides each contain modified amino acid residues, including derivatives of N-(1,1-dimethylallyl)-tryptophan and delta-hydroxyleucine, which are common in the di- and heptapeptide series. Stable isotope incorporation studies in Streptomyces sp. CNB-982, which was first reported to produce the cyclomarin anti-inflammatory agents, illuminated the biosynthetic building blocks associated with the major metabolite cyclomarin A, signifying that this marine microbial peptide is nonribosomally derived largely from nonproteinogenic amino acid residues. DNA sequence analysis of the 5.8 Mb S. arenicola circular genome and PCR-targeted gene inactivation experiments identified the 47 kb cyclomarin/cyclomarazine biosynthetic gene cluster (cym) harboring 23 open reading frames. The cym locus is dominated by the 23 358 bp cymA, which encodes a 7-module nonribosomal peptide synthetase (NRPS) responsible for assembly of the full-length cyclomarin heptapeptides as well as the truncated cyclomarazine dipeptides. The unprecedented biosynthetic feature of the megasynthetase CymA to synthesize differently sized peptides in vivo may be triggered by the level of beta oxidation of the priming tryptophan residue, which is oxidized in the cyclomarin series and unoxidized in the cyclomarazines. Biosynthesis of the N-(1,1-dimethyl-2,3-epoxypropyl)-beta-hydroxytryptophan residue of cyclomarin A was further illuminated through gene inactivation experiments, which suggest that the tryptophan residue is reverse prenylated by CymD prior to release of the cyclic peptide from the CymA megasynthetase, whereas the cytochrome P450 CymV installs the epoxide group on the isoprene of cyclomarin C post-NRPS assembly. Last, the novel amino acid residue 2-amino-3,5-dimethylhex-4-enoic acid in the cyclomarin series was shown by bioinformatics and stable isotope experiments to derive from a new pathway involving condensation of isobutyraldehyde and pyruvate followed by S-adenosylmethionine methylation. Assembly of this unsaturated, branched amino acid is unexpectedly related to the degradation of the environmental pollutant 3-(3-hydroxyphenyl)propionic acid.

A method for prediction of the locations of linker regions within large multifunctional proteins, and application to a type I polyketide synthase.

Multifunctional proteins often appear to result from fusion of smaller proteins and in such cases typically can be separated into their ancestral components simply by cleaving the linker regions that separate the domains. Though possibly guided by sequence alignment, structural evidence, or light proteolysis, determination of the locations of linker regions remains empirical. We have developed an algorithm, named UMA, to predict the locations of linker regions in multifunctional proteins by quantification of the conservation of several properties within protein families, and the results agree well with structurally characterized proteins. This technique has been applied to a family of fungal type I iterative polyketide synthases (PKS), allowing prediction of the locations of all of the standard PKS domains, as well as two previously unidentified domains. Using these predictions, we report the cloning of the first fragment from the PKS norsolorinic acid synthase, responsible for biosynthesis of the first isolatable intermediate in aflatoxin production. The expression, light proteolysis and catalytic abilities of this acyl carrier protein-thioesterase didomain are discussed.

Community annotation and bioinformatics workforce development in concert--Little Skate Genome Annotation Workshops and Jamborees.

Recent advances in high-throughput DNA sequencing technologies have equipped biologists with a powerful new set of tools for advancing research goals. The resulting flood of sequence data has made it critically important to train the next generation of scientists to handle the inherent bioinformatic challenges. The North East Bioinformatics Collaborative (NEBC) is undertaking the genome sequencing and annotation of the little skate (Leucoraja erinacea) to promote advancement of bioinformatics infrastructure in our region, with an emphasis on practical education to create a critical mass of informatically savvy life scientists. In support of the Little Skate Genome Project, the NEBC members have developed several annotation workshops and jamborees to provide training in genome sequencing, annotation and analysis. Acting as a nexus for both curation activities and dissemination of project data, a project web portal, SkateBase (http://skatebase.org) has been developed. As a case study to illustrate effective coupling of community annotation with workforce development, we report the results of the Mitochondrial Genome Annotation Jamborees organized to annotate the first completely assembled element of the Little Skate Genome Project, as a culminating experience for participants from our three prior annotation workshops. We are applying the physical/virtual infrastructure and lessons learned from these activities to enhance and streamline the genome annotation workflow, as we look toward our continuing efforts for larger-scale functional and structural community annotation of the L. erinacea genome.

Genomic islands link secondary metabolism to functional adaptation in marine Actinobacteria.

Genomic islands have been shown to harbor functional traits that differentiate ecologically distinct populations of environmental bacteria. A comparative analysis of the complete genome sequences of the marine Actinobacteria Salinispora tropica and Salinispora arenicola reveals that 75% of the species-specific genes are located in 21 genomic islands. These islands are enriched in genes associated with secondary metabolite biosynthesis providing evidence that secondary metabolism is linked to functional adaptation. Secondary metabolism accounts for 8.8% and 10.9% of the genes in the S. tropica and S. arenicola genomes, respectively, and represents the major functional category of annotated genes that differentiates the two species. Genomic islands harbor all 25 of the species-specific biosynthetic pathways, the majority of which occur in S. arenicola and may contribute to the cosmopolitan distribution of this species. Genome evolution is dominated by gene duplication and acquisition, which in the case of secondary metabolism provide immediate opportunities for the production of new bioactive products. Evidence that secondary metabolic pathways are exchanged horizontally, coupled with earlier evidence for fixation among globally distributed populations, supports a functional role and suggests that the acquisition of natural product biosynthetic gene clusters represents a previously unrecognized force driving bacterial diversification. Species-specific differences observed in clustered regularly interspaced short palindromic repeat sequences suggest that S. arenicola may possess a higher level of phage immunity, whereas a highly duplicated family of polymorphic membrane proteins provides evidence for a new mechanism of marine adaptation in Gram-positive bacteria.

Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica.

Recent fermentation studies have identified actinomycetes of the marine-dwelling genus Salinispora as prolific natural product producers. To further evaluate their biosynthetic potential, we sequenced the 5,183,331-bp S. tropica CNB-440 circular genome and analyzed all identifiable secondary natural product gene clusters. Our analysis shows that S. tropica dedicates a large percentage of its genome ( approximately 9.9%) to natural product assembly, which is greater than previous Streptomyces genome sequences as well as other natural product-producing actinomycetes. The S. tropica genome features polyketide synthase systems of every known formally classified family, nonribosomal peptide synthetases, and several hybrid clusters. Although a few clusters appear to encode molecules previously identified in Streptomyces species, the majority of the 17 biosynthetic loci are novel. Specific chemical information about putative and observed natural product molecules is presented and discussed. In addition, our bioinformatic analysis not only was critical for the structure elucidation of the polyene macrolactam salinilactam A, but its structural analysis aided the genome assembly of the highly repetitive slm loci. This study firmly establishes the genus Salinispora as a rich source of drug-like molecules and importantly reveals the powerful interplay between genomic analysis and traditional natural product isolation studies.

Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase.

Polyketides are a class of natural products that exhibit a wide range of functional and structural diversity. They include antibiotics, immunosuppressants, antifungals, antihypercholesterolemics, and cytotoxins. Polyketide synthases (PKSs) use chemistry similar to fatty acid synthases (FASs), although building block variation and differing extents of reduction of the growing polyketide chain underlie their biosynthetic versatility. In contrast to the well studied sequential modular type I PKSs, less is known about how the iterative type I PKSs carry out and control chain initiation, elongation, folding, and cyclization during polyketide processing. Domain structure analysis of a group of related fungal, nonreducing PKSs has revealed well defined N-terminal domains longer than commonly seen for FASs and modular PKSs. Predicted structure of this domain disclosed a region similar to malonyl-CoA:acyl-carrier protein (ACP) transacylases (MATs). MATs play a key role transferring precursor CoA thioesters from solution onto FASs and PKSs for chain elongation. On the basis of site-directed mutagenesis, radiolabeling, and kinetics experiments carried out with individual domains of the norsolorinic acid PKS, we propose that the N-terminal domain is a starter unit:ACP transacylase (SAT domain) that selects a C(6) fatty acid from a dedicated yeast-like FAS and transfers this unit onto the PKS ACP, leading to the production of the aflatoxin precursor, norsolorinic acid. These findings could indicate a much broader role for SAT domains in starter unit selection among nonreducing iterative, fungal PKSs, and they provide a biochemical rationale for the classical acetyl "starter unit effect."

Type III polyketide synthase beta-ketoacyl-ACP starter unit and ethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genome mining.

Polyketide synthases (PKSs) are involved in the biosynthesis of many important natural products. In bacteria, type III PKSs typically catalyze iterative decarboxylation and condensation reactions of malonyl-CoA building blocks in the biosynthesis of polyhydroxyaromatic products. Here it is shown that Gcs, a type III PKS encoded by the sco7221 ORF of the bacterium Streptomyces coelicolor, is required for biosynthesis of the germicidin family of 3,6-dialkyl-4-hydroxypyran-2-one natural products. Evidence consistent with Gcs-catalyzed elongation of specific beta-ketoacyl-ACP products of the fatty acid synthase FabH with ethyl- or methylmalonyl-CoA in the biosynthesis of germicidins is presented. Selectivity for beta-ketoacyl-ACP starter units and ethylmalonyl-CoA as an extender unit is unprecedented for type III PKSs, suggesting these enzymes may be capable of utilizing a far wider range of starter and extender units for natural product assembly than believed until now.

Synthesis of 11-hydroxyl O-methylsterigmatocystin and the role of a cytochrome P-450 in the final step of aflatoxin biosynthesis.

The major skeletal rearrangements (anthraquinone --> xanthone --> coumarin) that occur in the complex biosynthesis of aflatoxin B(1) are mediated by cytochromes P-450. Previous experiments have suggested that two successive monooxygenase reactions are required to convert the xanthone O-methylsterigmatocystin (OMST) to aflatoxin, a process we demonstrate is mediated by a single P-450, OrdA, in Aspergillus parasiticus in accord with findings in A. flavus. The first oxidative cycle is proposed to result in the formation of 11-hydroxy O-methylsterigmatocystin (HOMST), while the second entails aryl ring cleavage, demethylation, dehydration, decarboxylation, and rearrangement to give aflatoxin - a remarkable sequence of transformations. To test this hypothesis, HOMST has been synthesized by an alkylnitrilium variant of the Houben-Hoesch reaction. The troublesome xanthone carbonyl was protected as a butylene to allow further elaboration of the molecule, and then the product xanthone was restored in a uniquely facile peracid deprotection. Methods were devised to construct the sensitive dihydrobisfuran and to maintain the oxidation state of the partially methylated hydroquinone. Expression of ordA in a yeast membrane preparation enabled the intermediacy of HOMST both to be detected in the conversion of OMST to aflatoxin and to be established directly in the biosynthesis of the mycotoxin. Having secured the role of HOMST in aflatoxin formation, the mechanism of the second oxidative cycle of this P-450 is considered.

Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates.

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation.