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Agarwal, V, Diethelm S, Ray L, Garg N, Awakawa T, Dorrestein PC, Moore BS.  2015.  Chemoenzymatic synthesis of acyl coenzyme a substrates enables in situ labeling of small molecules and proteins. Organic Letters. 17:4452-4455.   10.1021/acs.orglett.5b02113   AbstractWebsite

A chemoenzymatic approach to generate fully functional acyl coenzyme A molecules that are then used as substrates to drive in situ acyl transfer reactions is described. Mass spectrometry based assays to verify the identity of acyl coenzyme A enzymatic products are also illustrated. The approach is responsive to a diverse array of carboxylic acids that can be elaborated to their corresponding coenzyme A thioesters, with potential applications in wide-ranging chemical biology studies that utilize acyl coenzyme A substrates.

Agarwal, V, Li J, Rahman I, Borgen M, Aluwihare LI, Biggs JS, Paul VJ, Moore BS.  2015.  Complexity of naturally produced polybrominated diphenyl ethers revealed via mass spectrometry. Environmental Science & Technology. 49:1339-1346.   10.1021/es505440j   AbstractWebsite

Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative anthropogenic and natural chemicals that are broadly distributed in the marine environment. PBDEs are potentially toxic due to inhibition of various mammalian signaling pathways and enzymatic reactions. PBDE isoforms vary in toxicity in accordance with structural differences, primarily in the number and pattern of hydroxyl moieties afforded upon a conserved core structure. Over four decades of isolation and discovery-based efforts have established an impressive repertoire of natural PBDEs. Based on our recent reports describing the bacterial biosyntheses of PBDEs, we predicted the presence of additional classes of PBDEs to those previously identified from marine sources. Using mass spectrometry and NMR spectroscopy, we now establish the existence of new structural classes of PBDEs in marine sponges. Our findings expand the chemical space explored by naturally produced PBDEs, which may inform future environmental toxicology studies. Furthermore, we provide evidence for iodinated PBDEs and direct attention toward the contribution of promiscuous halogenating enzymes in further expanding the diversity of these polyhalogenated marine natural products.

Agarwal, V, El Gamal AA, Yamanaka K, Poth D, Kersten RD, Schorn M, Allen EE, Moore BS.  2014.  Biosynthesis of polybrominated aromatic organic compounds by marine bacteria. Nature Chemical Biology. 10:640-U182.   10.1038/nchembio.1564   AbstractWebsite

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds.

Agarwal, V, Miles ZD, Winter JM, Eustaquio AS, El Gamal AA, Moore BS.  2017.  Enzymatic halogenation and dehalogenation reactions: Pervasive and mechanistically diverse. Chemical Reviews. 117:5619-5674.   10.1021/acs.chemrev.6b00571   AbstractWebsite

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.

Agarwal, V, Moore BS.  2014.  Enzymatic synthesis of polybrominated dioxins from the marine environment. Acs Chemical Biology. 9:1980-1984.   10.1021/cb5004338   AbstractWebsite

Polyhalogenated dibenzo-p-dioxins are arguably among the most toxic molecules known to man. In addition to anthropogenic sources, marine invertebrates also harbor polybrominated dibenzo-p-dioxins of as yet unknown biogenic origin. Here, we report that the bmp gene locus in marine bacteria, a recently characterized source of polybrominated diphenyl ethers, can also synthesize dibenzo-p-dioxins by employing different phenolic initiator molecules. Our findings also diversify the structural classes of diphenyl ethers accessed by the mmp biosynthetic pathway. This report lays the biochemical foundation of a likely biogenetic origin of dibenzo-p-dioxins present in the marine metabolome and greatly expands the toxicity potential of marine derived polyhaloganated natural products.

Agarwal, V, Blanton JM, Podell S, Taton A, Schorn MA, Busch J, Lin Z, Schmidt EW, Jensen PR, Paul VJ, Biggs JS, Golden JW, Allen EE, Moore BS.  2017.  Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges. Nat Chem Biol. advance online publication: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.   10.1038/nchembio.2330   Abstract

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.

Amos, GCA, Awakawa T, Tuttle RN, Letzel AC, Kim MC, Kudo Y, Fenical W, Moore BS, Jensen PR.  2017.  Comparative transcriptomics as a guide to natural product discovery and biosynthetic gene cluster functionality. Proceedings of the National Academy of Sciences of the United States of America. 114:E11121-E11130.   10.1073/pnas.1714381115   AbstractWebsite

Bacterial natural products remain an important source of new medicines. DNA sequencing has revealed that a majority of natural product biosynthetic gene clusters (BGCs) maintained in bacterial genomes have yet to be linked to the small molecules whose biosynthesis they encode. Efforts to discover the products of these orphan BGCs are driving the development of genome mining techniques based on the premise that many are transcriptionally silent during normal laboratory cultivation. Here, we employ comparative transcriptomics to assess BGC expression among four closely related strains of marine bacteria belonging to the genus Salinispora. The results reveal that slightly more than half of the BGCs are expressed at levels that should facilitate product detection. By comparing the expression profiles of similar gene clusters in different strains, we identified regulatory genes whose inactivation appears linked to cluster silencing. The significance of these subtle differences between expressed and silent BGCs could not have been predicted a priori and was only revealed by comparative transcriptomics. Evidence for the conservation of silent clusters among a larger number of strains for which genome sequences are available suggests they may be under different regulatory control from the expressed forms or that silencing may represent an underappreciated mechanism of gene cluster evolution. Coupling gene expression and metabolomics data established a bioinformatic link between the salinipostins and their associated BGC, while genetic manipulation established the genetic basis for this series of compounds, which were previously unknown from Salinispora pacifica.

Arnison, PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA, Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E, Donadio S, Dorrestein PC, Entian KD, Fischbach MA, Garavelli JS, Goransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Muller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJT, Rebuffat S, Ross RP, Sahl HG, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Sussmuth RD, Tagg JR, Tang GL, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, van der Donk WA.  2013.  Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Natural Product Reports. 30:108-160.   10.1039/c2np20085f   AbstractWebsite

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Austin, MB, Saito T, Bowman ME, Haydock S, Kato A, Moore BS, Kay RR, Noel JP.  2006.  Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid - type III polyketide synthase. Nature Chemical Biology. 2:494-502.   10.1038/nchembio811   AbstractWebsite

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two similar to 3,000-residue D. discoideum proteins, termed ` steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.

Austin, MB, Izumikawa M, Bowman ME, Udwary DW, Ferrer JL, Moore BS, Noel JP.  2004.  Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates. Journal of Biological Chemistry. 279:45162-45174.   10.1074/jbc.M406567200   AbstractWebsite

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene 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.

Awakawa, T, Crusemann M, Munguia J, Ziemert N, Nizet V, Fenical W, Moore BS.  2015.  Salinipyrone and pacificanone are biosynthetic by-products of the rosamicin polyketide synthase. Chembiochem. 16:1443-1447.   10.1002/cbic.201500177   AbstractWebsite

Salinipyrones and pacificanones are structurally related polyketides from Salinispora pacifica CNS-237 that are proposed to arise from the same modular polyketide synthase (PKS) assembly line. Genome sequencing revealed a large macrolide PKS gene cluster that codes for the biosynthesis of rosamicin A and a series of new macrolide antibiotics. Mutagenesis experiments unexpectedly correlated salinipyrone and pacificanone biosynthesis to the rosamicin octamodule Spr PKS. Remarkably, this bifurcated polyketide pathway illuminates a series of enzymatic domain- and module-skipping reactions that give rise to natural polyketide product diversity. Our findings enlarge the growing knowledge of polyketide biochemistry and illuminate potential challenges in PKS bioengineering.

Becker, JE, Moore RE, Moore BS.  2004.  Cloning, sequencing, and biochemical characterization of the nostocyclopeptide biosynthetic gene cluster: molecular basis for imine macrocyclization. Gene. 325:35-42.   10.1016/j.gene.2003.09.034   AbstractWebsite

Nostocyclopeptides A1 and A2 are novel cyclic heptapeptides produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789 that possess a unique imino linkage in the macrocyclic ring. Herein we report the cloning, sequencing, annotation, and biochemical analysis of the 33-kb nostocyclopeptide (ncp) biosynthetic gene cluster, which includes seven open reading frames predicted to be involved in the biosynthesis and transport of these natural products. The genetic architecture and domain organization of the ncpA-B nonribosomal peptide synthetase (NRPS) is co-linear in arrangement with respect to the putative order of the biosynthetic assembly of the cyclic peptide. A reductase domain identified at the C-terminal end of the NRPS NcpB is predicted to catalyze an NAD(P)H-mediated hydride transfer to the heptapeptidyl-S-enzyme intermediate NH2-Tyr-Gly-DGln-Ile-Ser-mPro-Leu/Phe-S-NRPS to yield a linear heptapeptide aldehyde that is subsequently captured intramolecularly with the amino group of the N-terminal amino acid residue tyrosine to form a stable imine bond. While a few C-terminal reductases associated with NRPPSs have been identified, the ncp reductase is the first to mediate imine macrocyclization involving peptide N- and C-termini. Biochemical analysis of the NcpA1 and NcpB1 adenylation domains coupled with the recent characterization of the (2S,4S)-5-hydroxyleucine dehydrogenase NcpD, which is involved in the biosynthesis of the nonproteinogenic amino acid residue L-4-methylproline from L-leucine, support the involvement of this cluster in nostocyclopeptide biosynthesis. (C) 2003 Elsevier B.V. All rights reserved.

Beer, LL, Moore BS.  2007.  Biosynthetic convergence of salinosporamides A and B in the marine actinomycete Salinispora tropica. Organic Letters. 9:845-848.   10.1021/ol063102o   AbstractWebsite

Feeding experiments with stable isotopes established that the potent 20S-proteasome inhibitors salinosporamide A and B are biosynthesized in the marine bacterium Salinispora tropica from three biosynthetic building blocks, namely, acetate, beta-hydroxy-2'-cyclohexenylalanine, and either butyrate or a tetrose-derived chlorinated molecule. The unexpected observation that the chlorinated four-carbon residue in salinosporamide A is derived from a different metabolic origin than the non-chlorinated four-carbon unit in salinosporamide B is suggestive of a convergent biosynthesis to these two anticancer natural products.

Bernhardt, P, Okino T, Winter JM, Miyanaga A, Moore BS.  2011.  A Stereoselective Vanadium-Dependent Chloroperoxidase in Bacterial Antibiotic Biosynthesis. Journal of the American Chemical Society. 133:4268-4270.   10.1021/ja201088k   AbstractWebsite

Halogenases catalyze reactions that introduce halogen atoms into electron-rich organic molecules. Vanadium-dependent haloperoxidases are generally considered to be promiscuous halogenating enzymes that have thus far been derived exclusively from eukaryotes, where their cellular function is often disputed. We now report the first biochemical characterization of a bacterial vanadium-dependent chloroperoxidase, NapH1 from Streptomyces sp. CNQ-525, which catalyzes a highly stereoselective chlorination-cyclization reaction in napyradiomycin antibiotic biosynthesis. This finding biochemically links a vanadium chloroperoxidase to microbial natural product biosynthesis.

Bonet, B, Teufel R, Crusemann M, Ziemert N, Moore BS.  2015.  Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin. Journal of Natural Products. 78:539-542.   10.1021/np500664q   AbstractWebsite

Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

Bruns, H, Crusemann M, Letzel AC, Alanjary M, McInerney JO, Jensen PR, Schulz S, Moore BS, Ziemert N.  2018.  Function-related replacement of bacterial siderophore pathways. Isme Journal. 12:320-329.   10.1038/ismej.2017.137   AbstractWebsite

Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co- occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters.

Brunson, JK, McKinnie SMK, Chekan JR, McCrow JP, Miles ZD, Bertrand EM, Bielinski VA, Luhavaya H, Oborník M, Smith JG, Hutchins DA, Allen AE, Moore BS.  2018.  Biosynthesis of the neurotoxin domoic acid in a bloom-forming diatom. Science. 361:1356-1358.   10.1126/science.aau0382   Abstract

Algal blooms can devastate marine mammal communities through the production of neurotoxins that accumulate within the food web. Brunson et al. identified a cluster of genes associated with biosynthesis of the neurotoxin domoic acid in a marine diatom (see the Perspective by Pohnert et al.). In vitro experiments established a series of enzymes that create the core structure of the toxin. Knowledge of the genes involved in domoic acid production will allow for genetic monitoring of algal blooms and aid in identifying conditions that trigger toxin production.Science, this issue p. 1356; see also p. 1308Oceanic harmful algal blooms of Pseudo-nitzschia diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth conditions known to induce DA production in Pseudo-nitzschia multiseries, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA’s diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production.

Cha, JY, Han S, Hong HJ, Cho H, Kim D, Kwon Y, Kwon SK, Crusemann M, Lee YB, Kim JF, Giaever G, Nislow C, Moore BS, Thomashow LS, Weller DM, Kwak YS.  2016.  Microbial and biochemical basis of a Fusarium wilt-suppressive soil. Isme Journal. 10:119-129.   10.1038/ismej.2015.95   AbstractWebsite

Crops lack genetic resistance to most necrotrophic pathogens. To compensate for this disadvantage, plants recruit antagonistic members of the soil microbiome to defend their roots against pathogens and other pests. The best examples of this microbially based defense of roots are observed in disease-suppressive soils in which suppressiveness is induced by continuously growing crops that are susceptible to a pathogen, but the molecular basis of most is poorly understood. Here we report the microbial characterization of a Korean soil with specific suppressiveness to Fusarium wilt of strawberry. In this soil, an attack on strawberry roots by Fusarium oxysporum results in a response by microbial defenders, of which members of the Actinobacteria appear to have a key role. We also identify Streptomyces genes responsible for the ribosomal synthesis of a novel heat-stable antifungal thiopeptide antibiotic inhibitory to F. oxysporum and the antibiotic's mode of action against fungal cell wall biosynthesis. Both classical-and community-oriented approaches were required to dissect this suppressive soil from the field to the molecular level, and the results highlight the role of natural antibiotics as weapons in the microbial warfare in the rhizosphere that is integral to plant health, vigor and development.

Chen, ZH, Wang BL, Kale AJ, Moore BS, Wang RW, Qing FL.  2012.  Coupling of sterically hindered aldehyde with fluorinated synthons: Stereoselective synthesis of fluorinated analogues of salinosporamide A. Journal of Fluorine Chemistry. 136:12-19.   10.1016/j.jfluchem.2012.01.003   AbstractWebsite

Salinosporamide A is an irreversible inhibitor of the beta-subunits of the 20S proteasome. Its C-5 cyclohexenyl moiety is the key to its affinity and potency as an anticancer agent. Here we describe the synthesis of C-5 difluoromethylated and trifluoromethylated analogues of salinosporamide A and their biological evaluation as proteasome inhibitors against purified yeast 20S proteasome. The synthetic strategy featured the stereoselective coupling reaction of sterically hindered aldehyde 3 with fluorinated organolithium reagents. (C) 2012 Elsevier B.V. All rights reserved.

Cheng, Q, Xiang L, Izumikawa M, Meluzzi D, Moore BS.  2007.  Enzymatic total synthesis of enterocin polyketides. Nature Chemical Biology. 3:557-558.   10.1038/nchembio.2007.22   AbstractWebsite

Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.

Crusemann, M, O'Neill EC, Larson CB, Melnik AV, Floros DJ, da Silva RR, Jensen PR, Dorrestein PC, Moore BS.  2017.  Prioritizing natural product diversity in a collection of 146 bacterial strains based on growth and extraction protocols. Journal of Natural Products. 80:588-597.   10.1021/acsjnatprod.6b00722   AbstractWebsite

In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions.

Deng, H, McMahon SA, Eustaquio AS, Moore BS, Naismith JH, O'Hagan D.  2009.  Mechanistic Insights into Water Activation in SAM Hydroxide Adenosyltransferase (duf-62). Chembiochem. 10:2455-2459.   10.1002/cbic.200900369   AbstractWebsite

The substrate analogue S-adenosyl-L-homocysteine (SAH) was co-crystallised with SAM hydroxide adenosyltransferase from Pyrococcus horikoshii. Of the two active site water molecules one appears to be structural and the other is a candidate for nucleophilic attack, to become the C5′ adenosyl hydroxyl group. The data support a mechanism in which the Arg–Asp ion pair is important for positioning both water molecules.

Diethelm, S, Teufel R, Kaysser L, Moore BS.  2014.  A multitasking vanadium-dependent chloroperoxidase as an inspiration for the chemical synthesis of the merochlorins. Angewandte Chemie-International Edition. 53:11023-11026.   10.1002/anie.201405696   AbstractWebsite

The vanadium-dependent chloroperoxidase Mcl24 was discovered to mediate a complex series of unprecedented transformations in the biosynthesis of the merochlorin meroterpenoid antibiotics. In particular, a site-selective naphthol chlorination is followed by an oxidative dearomatization/terpene cyclization sequence to build up the stereochemically complex carbon framework of the merochlorins in one step. Inspired by the enzyme reactivity, a chemical chlorination protocol paralleling the biocatalytic process was developed. These chemical studies led to the identification of previously overlooked merochlorin natural products.

Doi-Katayama, Y, Tang L, Park C, Yu TW, Moore BS, Floss HG, Hutchinson CR.  1999.  Biosynthesis of the Ansamycin Antibiotic Rifamycin: Polyketide Synthase Processes Multiple Polyketide Chains Simultaneously. Symposium on the Chemistry of Natural Products. 41:637-642., Japan Abstract

The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase(PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF demonstrats that the PKS operates in a processive manner. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta- through decaketide have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with 18O experiments suggests origin 8-OH groupe of 8-hydroxy-7,8-dihydronaphthoquinone. Inaction of the rifR gene that encodes a thioesterase II-like protein shows it is unlikely that the RifR thioesterase catalyzes their release to a major extent.

Duncan, KR, Crusemann M, Lechner A, Sarkar A, Li J, Ziemert N, Wang MX, Bandeira N, Moore BS, Dorrestein PC, Jensen PR.  2015.  Molecular networking and pattern-based genome mining improves discovery of biosynthetic gene clusters and their products from Salinispora species. Chemistry & Biology. 22:460-471.   10.1016/j.chembiol.2015.03.010   AbstractWebsite

Genome sequencing has revealed that bacteria contain many more biosynthetic gene clusters than predicted based on the number of secondary metabolites discovered to date. While this biosynthetic reservoir has fostered interest in new tools for natural product discovery, there remains a gap between gene cluster detection and compound discovery. Here we apply molecular networking and the new concept of pattern-based genome mining to 35 Salinispora strains, including 30 for which draft genome sequences were either available or obtained for this study. The results provide a method to simultaneously compare large numbers of complex microbial extracts, which facilitated the identification of media components, known compounds and their derivatives, and new compounds that could be prioritized for structure elucidation. These efforts revealed considerable metabolite diversity and led to several molecular family-gene cluster pairings, of which the quinomycin-type depsipeptide retimycin A was characterized and linked to gene cluster NRPS40 using pattern-based bioinformatic approaches.