RESUMEN
Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.
Asunto(s)
Péptidos Cíclicos , Péptidos , Animales , Péptidos/genética , Péptidos Cíclicos/genética , Secuencia de Aminoácidos , Vendajes , Señales de Clasificación de ProteínaRESUMEN
Artificial intelligence-driven advances in protein structure prediction in recent years have raised the question: has the protein structure-prediction problem been solved? Here, with a focus on nonglobular proteins, we highlight the many strengths and potential weaknesses of DeepMind's AlphaFold2 in the context of its biological and therapeutic applications. We summarize the subtleties associated with evaluation of AlphaFold2 model quality and reliability using the predicted local distance difference test (pLDDT) and predicted aligned error (PAE) values. We highlight various classes of proteins that AlphaFold2 can be applied to and the caveats involved. Concrete examples of how AlphaFold2 models can be integrated with experimental data in the form of small-angle X-ray scattering (SAXS), solution NMR, cryo-electron microscopy (cryo-EM) and X-ray diffraction are discussed. Finally, we highlight the need to move beyond structure prediction of rigid, static structural snapshots toward conformational ensembles and alternate biologically relevant states. The overarching theme is that careful consideration is due when using AlphaFold2-generated models to generate testable hypotheses and structural models, rather than treating predicted models as de facto ground truth structures.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Proteínas , Dispersión del Ángulo Pequeño , Proteínas/química , Difracción de Rayos X , Microscopía por Crioelectrón , Pliegue de Proteína , Inteligencia Artificial , HumanosRESUMEN
Microbulbifer is a genus of halophilic bacteria that are commonly detected in the commensal marine microbiomes. These bacteria have been recognized for their ability to degrade polysaccharides and other polymeric materials. Increasingly, Microbulbifer genomes indicate these bacteria to be an untapped reservoir for novel natural product discovery and biosynthetic novelty. In this review, we summarize the distribution of Microbulbifer bacteria, activities of the various polymer degrading enzymes that these bacteria produce, and an up-to-date summary of the natural products that have been isolated from Microbulbifer strains. We argue that these bacteria have been hiding in plain sight, and contemporary efforts into their genome and metabolome mining are going to lead to a proliferation of Microbulbifer-derived natural products in the future. We also describe, where possible, the ecological interactions of these bacteria in marine microbiomes.
RESUMEN
Bioorthogonal derivatization of peptides and proteins enables investigations into their biological function and allows for exploitation of their therapeutic potential, among other varied deliverables. Herein, we describe a marine halogenating enzyme-assisted bioconjugation strategy in which an N-terminal leader peptide guides bromination of a C-terminal Trp residue in genetically encoded peptides and proteins, setting up further Trp arylation by Suzuki-Miyaura reactions. The bromination and subsequent cross-coupling reactions are residue-specific and regiospecific for the indole-6 position, occur under mild aqueous conditions, and do not require any modification of other Trp residues in the substrate peptide and/or protein. Workflows described herein demonstrate the applicability of halogenating enzymes in bioorthogonal conjugation chemistry.
Asunto(s)
Péptidos , Proteínas , Péptidos/química , Estructura Molecular , Catálisis , Señales de Clasificación de ProteínaRESUMEN
Commensal bacteria associated with marine invertebrates are underappreciated sources of chemically novel natural products. Using mass spectrometry, we had previously detected the presence of peptidic natural products in obligate marine bacteria of the genus Microbulbifer cultured from marine sponges. In this report, the isolation and structural characterization of a panel of ureidohexapeptide natural products, termed the bulbiferamides, from Microbulbifer strains is reported wherein the tryptophan side chain indole participates in a macrocyclizing peptide bond formation. Genome sequencing identifies biosynthetic gene clusters encoding production of the bulbiferamides and implicates the involvement of a thioesterase in the indolic macrocycle formation. The structural diversity and widespread presence of bulbiferamides in commensal microbiomes of marine invertebrates point toward a possible ecological role for these natural products.
Asunto(s)
Productos Biológicos , Poríferos , Animales , Productos Biológicos/química , Bacterias/genética , Poríferos/microbiología , Organismos Acuáticos , Acilación , IndolesRESUMEN
Ureidopeptidic natural products possess a wide variety of favorable pharmacological properties. In addition, they have been shown to mediate core physiological functions in producer bacteria. Here, we report that similar ureidopeptidic natural products with conserved biosynthetic gene clusters are produced by different bacterial genera that coinhabit marine invertebrate microbiomes. We demonstrate that a Microbulbifer strain isolated from a marine sponge can produce two different classes of ureidopeptide natural products encoded by two different biosynthetic gene clusters that are positioned on the bacterial chromosome and on a plasmid. The plasmid encoded ureidopeptide natural products, which we term the pseudobulbiferamides (5-8), resemble the ureidopeptide natural products produced by Pseudovibrio, a different marine bacterial genus that is likewise present in marine sponge commensal microbiomes. Using imaging mass spectrometry, we find that the two classes of Microbulbifer-derived ureidopeptides occupy different physical spaces relative to the bacterial colony, perhaps implying different roles for these two compound classes in Microbulbifer physiology and environmental interactions.
Asunto(s)
Productos Biológicos , Poríferos , Animales , Productos Biológicos/química , Poríferos/química , Bacterias , Plásmidos/genética , Familia de Multigenes , FilogeniaRESUMEN
Sponges are the richest source of bioactive organic small molecules, referred to as natural products, in the marine environment. It is well established that laboratory culturing-resistant symbiotic bacteria residing within the eukaryotic sponge host matrix often synthesize the natural products that are detected in the sponge tissue extracts. However, the contributions of the culturing-amenable commensal bacteria that are also associated with the sponge host to the overall metabolome of the sponge holobiont are not well defined. In this study, we cultured a large library of bacteria from three marine sponges commonly found in the Florida Keys. Metabolomes of isolated bacterial strains and that of the sponge holobiont were compared using mass spectrometry to reveal minimal metabolomic overlap between commensal bacteria and the sponge hosts. We also find that the phylogenetic overlap between cultured commensal bacteria and that of the sponge microbiome is minimal. Despite these observations, the commensal bacteria were found to be a rich resource for novel natural product discovery. Mass spectrometry-based metabolomics provided structural insights into these cryptic natural products. Pedagogic innovation in the form of laboratory curricula development is described which provided undergraduate students with hands-on instruction in microbiology and natural product discovery using metabolomic data mining strategies.
Asunto(s)
Productos Biológicos , Poríferos , Animales , Humanos , Filogenia , Georgia , Poríferos/microbiología , Bacterias , Metabolómica , Estudiantes , Productos Biológicos/químicaRESUMEN
Thiotemplated pyrrole is a prevailing intermediate in the synthesis of numerous natural products in which the pyrrole is tethered to a carrier protein (CP). Biosynthesis of the pyrrole requires oxidation of an l-proline side chain. Herein, we investigate the biocatalytic mechanism of proline-to-pyrrole synthesis by molecular dynamics simulations, quantum mechanics/molecular mechanics simulations, and electronic structure calculations using the recently reported (Thapa, H. R., et al. Biochemistry 2019, 58, 918) structure of a type II nonribosomal protein synthetase (NRPS) Bmp3-Bmp1 (Oxidase-CP) complex. The substrate (l-proline) is attached to the Bmp1(CP), and the catalytic site is located inside the flavin-dependent oxidase (Bmp3). We show that the FAD isoalloxazine ring is stabilized in the catalytic site of Bmp3 by strong hydrogen bonding with Asn123, Ile125, Ser126, and Thr158. After the initial deprotonation followed by an enamine-imine tautomerization, oxidation of the C2-C3 or C2-N1 bond, through a hydride transfer (from either C3 or N1), is required for the pyrrole synthesis. Computational results indicate that the hydride transfer is more likely to occur from C3 than N1. Additionally, we demonstrate the elasticity in the oxidase active site through enzymatic synthesis of proline derivatives.
Asunto(s)
Prolina/química , Prolina/metabolismo , Pirroles/química , Pirroles/metabolismo , Biocatálisis , Proteína Morfogenética Ósea 3/metabolismo , Proteínas Portadoras/metabolismo , Dominio Catalítico , Flavinas/química , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Estructura Molecular , Oxidación-Reducción , Oxidorreductasas/metabolismo , Conformación Proteica , Teoría CuánticaRESUMEN
Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Juego de Reactivos para Diagnóstico/economía , SARS-CoV-2/genética , Transferencia de Tecnología , Universidades/economía , Biotecnología/métodos , COVID-19/virología , Humanos , Juego de Reactivos para Diagnóstico/provisión & distribución , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/aislamiento & purificaciónRESUMEN
Assembly line biosynthesis of polyketide natural products involves checkpoints where identities of thiotemplated intermediates are verified before polyketide extension reactions are allowed to proceed. Determining what these checkpoints are and how they operate is critical for reprogramming polyketide assembly lines. Here we demonstrate that ketosynthase (KS) domains can perform this gatekeeping role. By comparing the substrate specificities for polyketide synthases that extend pyrrolyl and halogenated pyrrolyl substrates, we find that KS domains that need to differentiate between these two substrates exercise high selectivity. We additionally find that amino acid residues in the KS active site facilitate this selectivity and that these residues are amenable to rational engineering. On the other hand, KS domains that do not need to make selectivity decisions in their native physiological context are substrate-promiscuous. We also provide evidence that delivery of substrates to polyketide synthases by non-native carrier proteins is accompanied by reduced biosynthetic efficiency.
Asunto(s)
Productos Biológicos/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Productos Biológicos/química , Sintasas Poliquetidas/química , Policétidos/químicaRESUMEN
Marine sponges are prolific sources of bioactive natural products, several of which are produced by bacteria symbiotically associated with the sponge host. Bacteria-derived natural products, and the specialized bacterial symbionts that synthesize them, are not shared among phylogenetically distant sponge hosts. This is in contrast to nonsymbiotic culturable bacteria in which the conservation of natural products and natural product biosynthetic gene clusters (BGCs) is well established. Here, we demonstrate the widespread conservation of a BGC encoding a cryptic ribosomally synthesized and post-translationally modified peptide (RiPP) in microbiomes of phylogenetically and geographically dispersed sponges from the Pacific and Atlantic oceans. Detection of this BGC was enabled by mining for halogenating enzymes in sponge metagenomes, which, in turn, allowed for the description of a broad-spectrum regiospecific peptidyl tryptophan-6-brominase which possessed no chlorination activity. In addition, we demonstrate the cyclodehydrative installation of azoline heterocycles in proteusin RiPPs. This is the first demonstration of halogenation and cyclodehydration for proteusin RiPPs and the enzymes catalyzing these transformations were found to competently interact with other previously described proteusin substrate peptides. Within a sponge microbiome, many different generalized bacterial taxa harbored this BGC with often more than 50 copies of the BGC detected in individual sponge metagenomes. Moreover, the BGC was found in all sponges queried that possess high diversity microbiomes but it was not detected in other marine invertebrate microbiomes. These data shed light on conservation of cryptic natural product biosynthetic potential in marine sponges that was not detected by traditional natural product-to-BGC (meta)genome mining.
Asunto(s)
Bacterias/enzimología , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Microbiota/fisiología , Poríferos/microbiología , Secuencia de Aminoácidos , Animales , Productos Biológicos , Metagenoma , Familia de MultigenesRESUMEN
Proline-rich macrocyclic peptides (PRMPs) are natural products present in geographically and phylogenetically dispersed marine sponges. The large diversity and low abundance of PRMPs in sponge metabolomes precludes isolation and structure elucidation of each individual PRMP congener. Here, using standards developed via biomimetic enzymatic synthesis of PRMPs, a mass spectrometry-based workflow to sequence PRMPs was developed and validated to reveal that the diversity of PRMPs in marine sponges is much greater than that has been realized by natural product isolation-based strategies. Findings are placed in the context of diversity-oriented transamidative macrocyclization of peptide substrates in sponge holobionts.
Asunto(s)
Poríferos , AnimalesRESUMEN
Marine algae are prolific producers of bromoform (CHBr3 ). This naturally produced molecule is a potent environmental pollutant as it volatilizes into the atmosphere and contributes to depletion of the ozone layer in a manner akin to, and in magnitude similar to, man-made chlorofluorocarbons. While phototrophs such as seaweeds, diatoms, and dinoflagellates are known sources of bromoform, additional as yet unknown biogenetic sources of bromoform exist in the oceans. Here, using halogenating enzymes as diagnostic genetic elements, we demonstrate that marine cyanobacteria also possess the enzymological potential for bromoform production. Using recombinantly purified vanadium-dependent bromoperoxidases from planktonic and bloom-forming marine cyanobacteria in in vitro biochemical assays, we reconstitute the enzymatic production of bromoform. We find cyanobacterial bromoform synthesizing enzymes to be obligate brominases possessing no chlorinating activities. These results expand the repertoire of marine biotic sources that introduce this pollutant in the atmosphere.
Asunto(s)
Cianobacterias , Diatomeas , Océanos y Mares , TrihalometanosRESUMEN
Marine sponge holobionts, defined as filter-feeding sponge hosts together with their associated microbiomes, are prolific sources of natural products. The inventory of natural products that have been isolated from marine sponges is extensive. Here, using untargeted mass spectrometry, we demonstrate that sponges harbor a far greater diversity of low-abundance natural products that have evaded discovery. While these low-abundance natural products may not be feasible to isolate, insights into their chemical structures can be gleaned by careful curation of mass fragmentation spectra. Sponges are also some of the most complex, multi-organismal holobiont communities in the oceans. We overlay sponge metabolomes with their microbiome structures and detailed metagenomic characterization to discover candidate gene clusters that encode production of sponge-derived natural products. The multi-omic profiling strategy for sponges that we describe here enables quantitative comparison of sponge metabolomes and microbiomes to address, among other questions, the ecological relevance of sponge natural products and for the phylochemical assignment of previously undescribed sponge identities.
Asunto(s)
Ecosistema , Metabolómica , Microbiota , Poríferos/metabolismo , Poríferos/microbiología , Animales , Filogenia , Poríferos/genéticaRESUMEN
Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Pirroles/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Dominio Catalítico , Cristalografía por Rayos X , Dinitrocresoles/metabolismo , Marinomonas/genética , Marinomonas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Oxidorreductasas/genética , Conformación Proteica , Pirroles/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The production of pentabromopseudilin and related brominated compounds by Pseudoalteromonas spp. has recently been linked to the bmp biosynthetic gene cluster. This study explored the distribution and evolutionary history of this gene cluster in the genus Pseudoalteromonas. A phylogeny of the genus revealed numerous clades that do not contain type strains, suggesting considerable species level diversity has yet to be described. Comparative genomics revealed four distinct versions of the gene cluster distributed among 19 of the 101 Pseudoalteromonas genomes examined. These were largely localized to the least inclusive clades containing the Pseudoalteromonas luteoviolacea and Pseudoalteromonas phenolica type strains and show clear evidence of gene and gene cluster loss in certain lineages. Bmp gene phylogeny is largely congruent with the Pseudoalteromonas species phylogeny, suggesting vertical inheritance within the genus. However, the gene cluster is found in three different genomic environments suggesting either chromosomal rearrangement or multiple acquisition events. Bmp conservation within certain lineages suggests the encoded products are highly relevant to the ecology of these bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Familia de Multigenes , Pseudoalteromonas/genética , Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Genómica , Filogenia , Pseudoalteromonas/clasificación , Pseudoalteromonas/aislamiento & purificación , Pseudoalteromonas/metabolismo , Pirroles/química , Pirroles/metabolismoRESUMEN
Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.
Asunto(s)
Adenosina Monofosfato/metabolismo , Coenzima A Ligasas/química , Modelos Moleculares , Adenosina Monofosfato/química , Dominio Catalítico , Coenzima A Ligasas/metabolismo , Cristalización , Ligandos , Estructura Molecular , Especificidad por SustratoRESUMEN
Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.
Asunto(s)
Productos Biológicos/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Éteres Difenilos Halogenados/metabolismo , Metagenómica , Poríferos/metabolismo , Animales , Productos Biológicos/química , Éteres Difenilos Halogenados/química , Estructura MolecularRESUMEN
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.
Asunto(s)
Enzimas/química , Halógenos/química , Flavinas/química , Vanadio/químicaRESUMEN
The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.