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Luhavaya, H, Sigrist R, Chekan JR, McKinnie SMK, Moore BS.  2019.  Biosynthesis of l-4-chlorokynurenine, an antidepressant prodrug and a non-proteinogenic amino acid found in lipopeptide antibiotics. Angewandte Chemie-International Edition. 58:8394-8399.   10.1002/anie.201901571   AbstractWebsite

l-4-Chlorokynurenine (l-4-Cl-Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish l-4-Cl-Kyn biosynthesis, which is initiated by the flavin-dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.

Schorn, MA, Jordan PA, Podell S, Blanton JM, Agarwal V, Biggs JS, Allen EE, Moore BS.  2019.  Comparative genomics of cyanobacterial symbionts reveals distinct, specialized metabolism in tropical Dysideidae sponges. mBio. 10( McFall-Ngai MJ, Donia M, Thacker R, Eds.). Abstract

Marine sponges are recognized as valuable sources of bioactive metabolites and renowned as petri dishes of the sea, providing specialized niches for many symbiotic microorganisms. Sponges of the family Dysideidae are well documented to be chemically talented, often containing high levels of polyhalogenated compounds, terpenoids, peptides, and other classes of bioactive small molecules. This group of tropical sponges hosts a high abundance of an uncultured filamentous cyanobacterium, Hormoscilla spongeliae. Here, we report the comparative genomic analyses of two phylogenetically distinct Hormoscilla populations, which reveal shared deficiencies in essential pathways, hinting at possible reasons for their uncultivable status, as well as differing biosynthetic machinery for the production of specialized metabolites. One symbiont population contains clustered genes for expanded polybrominated diphenylether (PBDE) biosynthesis, while the other instead harbors a unique gene cluster for the biosynthesis of the dysinosin nonribosomal peptides. The hybrid sequencing and assembly approach utilized here allows, for the first time, a comprehensive look into the genomes of these elusive sponge symbionts.IMPORTANCE Natural products provide the inspiration for most clinical drugs. With the rise in antibiotic resistance, it is imperative to discover new sources of chemical diversity. Bacteria living in symbiosis with marine invertebrates have emerged as an untapped source of natural chemistry. While symbiotic bacteria are often recalcitrant to growth in the lab, advances in metagenomic sequencing and assembly now make it possible to access their genetic blueprint. A cell enrichment procedure, combined with a hybrid sequencing and assembly approach, enabled detailed genomic analysis of uncultivated cyanobacterial symbiont populations in two chemically rich tropical marine sponges. These population genomes reveal a wealth of secondary metabolism potential as well as possible reasons for historical difficulties in their cultivation.

Busch, J, Agarwal V, Schorn M, Machado H, Moore BS, Rouse GW, Gram L, Jensen PR.  2019.  Diversity and distribution of the bmp gene cluster and its Polybrominated products in the genus Pseudoalteromonas. Environmental Microbiology. 21:1575-1585.   10.1111/1462-2920.14532   AbstractWebsite

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.

Walsh, CT, Moore BS.  2019.  Enzymatic cascade reactions in biosynthesis. Angewandte Chemie-International Edition. 58:6846-6879.   10.1002/anie.201807844   AbstractWebsite

Enzyme-mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron-based enzymes use O-2 to generate high valent iron-oxo species to homolyze unactivated C-H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S-adenosylmethionine (SAM) to generate the 5 '-deoxyadenosyl radical as a powerful oxidant to initiate C-H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as thwarted oxygenases.

Bauman, KD, Li J, Murata K, Mantovani SM, Dahesh S, Nizet V, Luhavaya H, Moore BS.  2019.  Refactoring the cryptic streptophenazine biosynthetic gene cluster unites phenazine, polyketide, and nonribosomal peptide biochemistry. Cell Chemical Biology. 26:724-+.   10.1016/j.chembiol.2019.02.004   AbstractWebsite

The disconnect between the genomic prediction of secondary metabolite biosynthetic potential and the observed laboratory production profile of microorganisms is well documented. While heterologous expression of biosynthetic gene clusters (BGCs) is often seen as a potential solution to bridge this gap, it is not immune to many challenges including impaired regulation, the inability to recruit essential building blocks, and transcriptional and/or translational silence of the biosynthetic genes. Here we report the discovery, cloning, refactoring, and heterologous expression of a cryptic hybrid phenazine-type BGC (spz) from the marine actinomycete Streptomyces sp. CNB-091. Overexpression of the engineered spz pathway resulted in increased production and chemical diversity of phenazine natural products belonging to the streptophenazine family, including bioactive members containing an unprecedented N-formylglycine attachment. An atypical discrete adenylation enzyme in the spz cluster is required to introduce the formylglycine moiety and represents a phylogenetically distinct class of adenylation proteins.

Rothe, ML, Li J, Garibay E, Moore BS, McKinnie SMK.  2019.  Synthesis, bioactivity, and enzymatic modification of antibacterial thiotetromycin derivatives. Organic & Biomolecular Chemistry. 17:3416-3423.   10.1039/c8ob03109f   AbstractWebsite

Thiotetronate-containing natural products, including thiolactomycin, thiotetromycin, and thiotetroamide, are potent, broad-spectrum antibacterial compounds that target fatty acid synthesis in bacteria. Natural modifications at the C-5 dialkyl position in this molecular series result in pronounced bioactivity differences. The C-5 acetamide-containing thiotetroamide, which is the more potent antibacterial agent in this family, is biosynthesized from the C-5 ethyl analogue thiotetromycin via a unique two-enzyme process involving the cytochrome P450-amidotransferase enzyme pair TtmP-TtmN. Herein we synthesized a focused library of 17 novel thiotetromycin derivatives differing at the 5-position alkyl substituent to investigate their biological activities and their reactivity towards the hydroxylase TtmP. Although we observed marginal anti-tuberculosis activity, select thiotetromycin analogues showed antibacterial activity against an Escherichia coli Delta tolC strain with IC50 values in a range of 1.9-36 mu g mL(-1). Additional screening efforts highlighted select thiotetronate analogues as inhibitors of the cancer-associated enzyme nicotinamide N-methyltransferase (NNMT), with a unique scaffold compared to previously identified NNMT inhibitors. In vitro assays further showed that the TtmP P450 was capable of resolving racemic substrate mixtures and had modest promiscuity to hydroxylate derivatives with variable alkyl chains; however triple oxidation to a carboxylic acid remained specific for the natural thiotetromycin substrate. The tendency of TtmP to accept a range of unnatural substrates for hydroxylation makes it an interesting target for P450 engineering towards broader applications.

Thapa, HR, Robbins JM, Moore BS, Agarwal V.  2019.  Insights into thiotemplated pyrrole biosynthesis gained from the crystal structure of flavin-dependent oxidase in complex with carrier protein. Biochemistry. 58:918-929.   10.1021/acs.biochem.8b01177   AbstractWebsite

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.

McAlpine, JB, Chen SN, Kutateladze A, MacMillan JB, Appendino G, Barison A, Beniddir MA, Biavatti MW, Bluml S, Boufridi A, Butler MS, Capon RJ, Choi YH, Coppage D, Crews P, Crimmins MT, Csete M, Dewapriya P, Egan JM, Garson MJ, Genta-Jouve G, Gerwick WH, Gross H, Harper MK, Hermanto P, Hook JM, Hunter L, Jeannerat D, Ji NY, Johnson TA, Kingston DGI, Koshino H, Lee HW, Lewin G, Li J, Linington RG, Liu MM, McPhail KL, Molinski TF, Moore BS, Nam JW, Neupane RP, Niemitz M, Nuzillard JM, Oberlies NH, Ocampos FMM, Pan G, Quinn RJ, Reddy DS, Renault JH, Rivera-Chavez J, Robien W, Saunders CM, Schmidt TJ, Seger C, Shen B, Steinbeck C, Stuppner H, Sturm S, Taglialatela-Scafati O, Tantillo DJ, Verpoorte R, Wang BG, Williams CM, Williams PG, Wist J, Yue JM, Zhang C, Xu ZR, Simmler C, Lankin DC, Bisson J, Pauli GF.  2019.  The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research. Natural Product Reports. 36:35-107.   10.1039/c7np00064b   AbstractWebsite

With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.

McKinnie, SMK, Miles ZD, Jordan PA, Awakawa T, Pepper HP, Murray LAM, George JH, Moore BS.  2018.  Total enzyme syntheses of napyradiomycins A1 and B1. Journal of the American Chemical Society. 140:17840-17845.   10.1021/jacs.8b10134   AbstractWebsite

The biosynthetic route to the napyradiomycin family of bacterial meroterpenoids has been fully described 32 years following their original isolation and 11 years after their gene cluster discovery. The antimicrobial and cytotoxic natural products napyradiomycins A1 and B1 are produced using three organic substrates (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), and catalysis via five enzymes: two aromatic prenyltransferases (NapT8 and T9); and three vanadium dependent haloperoxidase (VHPO) homologues (NapH1, H3, and H4). Building upon the previous characterization of NapH1, H3, and T8, we herein describe the initial (NapT9, H1) and final (NapH4) steps required for napyradiomycin construction. This remarkably streamlined biosynthesis highlights the utility of VHPO enzymology in complex natural product generation, as NapH4 efficiently performs a unique chloronium-induced terpenoid cyclization to establish two stereocenters and a new carbon-carbon bond, and dual-acting NapH1 catalyzes chlorination and etherification reactions at two distinct stages of the pathway. Moreover, we employed recombinant napyradiomycin biosynthetic enzymes to chemoenzymatically synthesize milligram quantities in one pot in 1 day. This method represents a viable enantioselective approach to produce complex halogenated metabolites, like napyradiomycin B1, that have yet to be chemically synthesized.

Schlawis, C, Kern S, Kudo Y, Grunenberg J, Moore BS, Schulz S.  2018.  Structural elucidation of trace components combining GC/MS, GC/IR, DFT-calculation and synthesis-salinilactones, unprecedented bicyclic lactones from Salinispora bacteria. Angewandte Chemie-International Edition. 57:14921-14925.   10.1002/anie.201807923   AbstractWebsite

The analysis of volatiles released by marine Salinispora bacteria uncovered a new class of natural compounds displaying an unusual bicyclic [3.1.0]-lactone skeleton. Although only sub-mu g quantities of the compounds were available, the combination of analytical methods, computational spectroscopy, and synthesis allowed unambiguous structural identification of the compounds, called salinilactones, without the need for isolation. Orthogonal hyphenated methods, GC/MS and solid-phase GC/IR allowed to propose a small set of structures consistent with the data. A candidate structure was selected by comparison of DFT-calculated IR spectra and the experimental IR-spectrum. Synthesis confirmed the structure and absolute configuration of three bicyclic lactones, salinilactones A-C. The salinilactones are structurally closely related to the A-factor class of compounds, autoregulators from streptomycete bacteria. They exhibited inhibitory activity against Salinispora and Streptomyces strains.

Whalen, KE, Kirby C, Nicholson RM, O'Reilly M, Moore BS, Harvey EL.  2018.  The chemical cue tetrabromopyrrole induces rapid cellular stress and mortality in phytoplankton. Scientific Reports. 8   10.1038/s41598-018-33945-3   AbstractWebsite

Eukaryotic phytoplankton contribute to the flow of elements through marine food webs, biogeochemical cycles, and Earth's climate. Therefore, how phytoplankton die is a critical determinate of the flow and fate of nutrients. While heterotroph grazing and viral infection contribute to phytoplankton mortality, recent evidence suggests that bacteria-derived cues also control phytoplankton lysis. Here, we report exposure to nanomolar concentrations of 2,3,4,5-tetrabromopyrrole (TBP), a brominated chemical cue synthesized by marine gamma-proteobacteria, resulted in mortality of seven phylogenetically-diverse phytoplankton species. A comparison of nine compounds of marine-origin containing a range of cyclic moieties and halogenation indicated that both a single pyrrole ring and increased bromination were most lethal to the coccolithophore, Emiliania huxleyi. TBP also rapidly induced the production of reactive oxygen species and the release of intracellular calcium stores, both of which can trigger the activation of cellular death pathways. Mining of the Ocean Gene Atlas indicated that TBP biosynthetic machinery is globally distributed throughout the water column in coastal areas. These findings suggest that bacterial cues play multiple functions in regulating phytoplankton communities by inducing biochemical changes associated with cellular death. Chemically-induced lysis by bacterial infochemicals is yet another variable that must be considered when modeling oceanic nutrient dynamics.

Zhang, JJ, Moore BS, Tang XY.  2018.  Engineering Salinispora tropica for heterologous expression of natural product biosynthetic gene clusters. Applied Microbiology and Biotechnology. 102:8437-8446.   10.1007/s00253-018-9283-z   AbstractWebsite

The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor Phi C31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.

Podell, S, Blanton JM, Neu A, Agarwal V, Biggs JS, Moore BS, Allen EE.  2018.  Pangenomic comparison of globally distributed Poribacteria associated with sponge hosts and marine particles. The ISME Journal.   10.1038/s41396-018-0292-9   AbstractWebsite

Candidatus Poribacteria is a little-known bacterial phylum, previously characterized by partial genomes from a single sponge host, but never isolated in culture. We have reconstructed multiple genome sequences from four different sponge genera and compared them to recently reported, uncharacterized Poribacteria genomes from the open ocean, discovering shared and unique functional characteristics. Two distinct, habitat-linked taxonomic lineages were identified, designated Entoporibacteria (sponge-associated) and Pelagiporibacteria (free-living). These lineages differed in flagellar motility and chemotaxis genes unique to Pelagiporibacteria, and highly expanded families of restriction endonucleases, DNA methylases, transposases, CRISPR repeats, and toxin–antitoxin gene pairs in Entoporibacteria. Both lineages shared pathways for facultative anaerobic metabolism, denitrification, fermentation, organosulfur compound utilization, type IV pili, cellulosomes, and bacterial proteosomes. Unexpectedly, many features characteristic of eukaryotic host association were also shared, including genes encoding the synthesis of eukaryotic-like cell adhesion molecules, extracellular matrix digestive enzymes, phosphoinositol-linked membrane glycolipids, and exopolysaccharide capsules. Complete Poribacteria 16S rRNA gene sequences were found to contain multiple mismatches to “universal” 16S rRNA gene primer sets, substantiating concerns about potential amplification failures in previous studies. A newly designed primer set corrects these mismatches, enabling more accurate assessment of Poribacteria abundance in diverse marine habitats where it may have previously been overlooked.

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.

Murray, LAM, McKinnie SMK, Pepper HP, Erni R, Miles ZD, Cruickshank MC, Lopez-Perez B, Moore BS, George JH.  2018.  Total synthesis establishes the biosynthetic pathway to the naphterpin and marinone natural products. Angewandte Chemie-International Edition. 57:11009-11014.   10.1002/anie.201804351   AbstractWebsite

The naphterpins and marinones are naphthoquinone meroterpenoids with an unusual aromatic oxidation pattern that is biosynthesized from 1,3,6,8-tetrahydroxynaphthalene (THN). We propose that cryptic halogenation of THN derivatives by vanadium-dependent chloroperoxidase (VCPO) enzymes is key to this biosynthetic pathway, despite the absence of chlorine in these natural products. This speculation inspired a total synthesis to mimic the naphterpin/marinone biosynthetic pathway. In validation of this biogenetic hypothesis, two VCPOs were discovered that interconvert several of the proposed biosynthetic intermediates.

Saleem-Batcha, R, Stull F, Sanders JN, Moore BS, Palfey BA, Houk KN, Teufel R.  2018.  Enzymatic control of dioxygen binding and functionalization of the flavin cofactor. Proceedings of the National Academy of Sciences of the United States of America. 115:4909-4914.   10.1073/pnas.1801189115   AbstractWebsite

The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O-2) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O-2, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O-2-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O-2 within a flavoenzyme's active site enables the regio-specific formation of a covalent flavin-oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O-2 functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O-2 reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O-2-reactive enzymes.

Zheng, J, McKinnie SMK, El Gama A, Fengit W, Dong Y, Agarwal V, Fenical W, Kumar A, Cao ZY, Moore BS, Pessah IN.  2018.  Organohalogens naturally biosynthesized in marine environments and produced as disinfection byproducts alter sarco/endoplasmic reticulum ca2+ dynamics. Environmental Science & Technology. 52:5469-5478.   10.1021/acs.est.8b00512   AbstractWebsite

Contemporary sources of organohalogens produced as disinfection byproducts (DBPs) are receiving considerable attention as emerging pollutants because of their abundance, persistence, and potential to structurally mimic natural organohalogens produced by bacteria that serve signaling or toxicological functions in marine environments. Here, we tested 34 organohalogens from anthropogenic and marine sources to identify compounds active toward ryanodine receptor (RyR1), known toxicological targets of non-dioxin like polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). [H-3]Ryanodine ([H-3]Ry) binding screening (<= 2 mu M) identified 10 highly active organohalogens. Further analysis indicated that 2,3-dibromoindole (14), tetrabromopyrrole (31), and 2,3,S-tribromopyrrole (34) at 10 mu M were the most efficacious at enhancing [H-3]Ry binding. Interestingly, these congeners also inhibited microsomal sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase (SERCA1a). Dual SERCAla inhibition and RyR1 activation triggered Ca2+ efflux from microsomal vesicles with initial rates rank ordered 31 > 34 > 14. Hexabromobipyrroles (25) enhanced [H-3]Ry binding moderately with strong SERCAla inhibition, whereas pyrrole (24), 2,3,4-tribromopyrrole (26), and ethyl-4-bromopyrrole-2-carboxylate (27) were inactive. Of three PBDE derivatives of marine origin active in the [H-3]Ry assay, 4'-hydroxy-2,3',4,5',6-pentabromodiphenyl ether (18) was also a highly potent SERCAla inhibitor. Molecular targets of marine organohalogens that are also DBPs of emerging environmental concern are likely to contribute to their toxicity.

Moore, BS.  2018.  Asymmetric Alkene and Arene Halofunctionalization Reactions in Meroterpenoid Biosynthesis. Synlett. 29:401-409.   10.1055/s-0036-1590919   AbstractWebsite

Meroterpenoid natural products are important bioactive molecules with broad distribution throughout nature. In Streptomyces bacteria, naphthoquinone-based meroterpenoids comprise a simple yet structurally fascinating group of natural product antibiotics that are enzymatically constructed through a series of asymmetric alkene and arene halofunctionalization reactions. This account article highlights our discovery and characterization of a group of vanadium-dependent chloroperoxidase enzymes that catalyze halogen-assisted cyclization and rearrangement reactions and have inspired biomimetic syntheses of numerous meroterpenoid natural products. 1 Introduction 2 Early Biosynthetic Insights and the Characterization of Alkene Halofunctionalization in Napyradiomycin Biosynthesis 3 Discovery of the Merochlorin Natural Products and Enzymatic Aryl Halofunctionalization 4 Discovery and Development of Unifying THN-Based Meroterpenoid Biosynthesis and Synthesis Approaches 5 Insights into Naphterpin and Marinone Biosynthesis Involving Cryptic Aryl Halofunctionalization Reactions 6 Closing Thoughts

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.

Reynolds, KA, Luhavaya H, Li J, Dahesh S, Nizet V, Yamanaka K, Moore BS.  2018.  Isolation and structure elucidation of lipopeptide antibiotic taromycin B from the activated taromycin biosynthetic gene cluster. Journal of Antibiotics. 71:333-338.   10.1038/ja.2017.146   AbstractWebsite

In the ongoing effort to unlock the chemical potential of marine bacteria, genetic engineering of biosynthetic gene clusters (BGCs) is increasingly used to awake or improve expression of biosynthetic genes that may lead to discovery of novel bioactive natural products. Previously, we reported the successful capture, engineering and heterologous expression of an orphan BGC from the marine actinomycete Saccharomonospora sp. CNQ-490, which resulted in the isolation of the novel lipopeptide antibiotic taromycin A. Herein we report the isolation and structure elucidation of taromycin B, the second most abundant product of the taromycin biosynthetic series, and show that taromycins A and B exhibit complex chromatographic properties indicative of interconverting conformations. Taromycins A and B display potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium clinical isolates, suggestive that the taromycin molecular scaffold is a promising starting point for further derivatization to produce compounds with promising antibiotic characteristics.

McKinnie, SMK, Miles ZD, Moore BS.  2018.  Characterization and biochemical assays of Streptomyces vanadium-dependent chloroperoxidases. Marine Enzymes and Specialized Metabolism, Pt A. 604( Moore BS, Ed.).:405-424., San Diego: Elsevier Academic Press Inc   10.1016/bs.mie.2018.02.016   Abstract

Vanadium-dependent haloperoxidases (VHPOs) are fascinating enzymes that facilitate electrophilic halogen incorporation into electron-rich substrates, simply requiring vanadate, a halide source, and cosubstrate hydrogen peroxide for activity. Initially characterized in fungi and red algae, VHPOs were long believed to have limited regio-, chemo-, and enantioselectivity in the production of halogenated metabolites. However, the recent discovery of homologues in the biosynthetic gene clusters of the stereo-selectively halogenated meroterpenoids from marine-derived Streptomyces bacteria has revised this paradigm. Their intriguing transformations have both enhanced and contributed to the fields of synthetic organic and natural product chemistry. We, herein, describe the expression, purification, and chemical assays of two characterized vanadium-dependent chloroperoxidase enzymes (NapH1 and Mcl24), and one homologue devoid of chlorination activity (NapH3), involved in the biosyntheses of halogenated meroterpenoid products.

Chekan, JR, Moore BS.  2018.  Preparation and characterization of tetrabromopyrrole debrominase from marine proteobacteria. Marine Enzymes and Specialized Metabolism, Pt B. 605( Moore BS, Ed.).:253-265., San Diego: Elsevier Academic Press Inc   10.1016/bs.mie.2018.01.031   Abstract

While halogenases have been studied for decades, the first natural product dehalogenase was only recently described. This bacterial enzyme, Bmp8, catalyzes the reductive debromination of 2,3,4,5-tetrabromopyrrole to form 2,3,4-tribromopyrrole as part of the biosynthesis of pentabromopseudilin, a marine natural product. Bmp8 is hypothesized to utilize a catalytic mechanism analogous to the important human thyroid hormone deiodinase enzyme family, potentially enabling Bmp8 to serve as model system to study this conserved mechanism. Herein, we describe a method for the soluble expression and purification of Bmp8. Furthermore, we detail activity assay protocols to quantify both consumption of the tetrabromopyrrole substrate and formation of the tribromopyrrole product. These methods will enable further study of this unusual enzyme and its catalytic mechanism.

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.

Miles, ZD, Diethelm S, Pepper HP, Huang DM, George JH, Moore BS.  2017.  A unifying paradigm for naphthoquinone-based meroterpenoid (bio)synthesis. Nature Chemistry. 9:1235-1242.   10.1038/nchem.2829   AbstractWebsite

Bacterial meroterpenoids constitute an important class of natural products with diverse biological properties and therapeutic potential. The biosynthetic logic for their production is unknown and defies explanation via classical biochemical paradigms. A large subgroup of naphthoquinone-based meroterpenoids exhibits a substitution pattern of the polyketide-derived aromatic core that seemingly contradicts the established reactivity pattern of polyketide phenol nucleophiles and terpene diphosphate electrophiles. We report the discovery of a hitherto unprecedented enzyme-promoted alpha-hydroxyketone rearrangement catalysed by vanadium-dependent haloperoxidases to account for these discrepancies in the merochlorin and napyradiomycin class of meroterpenoid antibiotics, and we demonstrate that the alpha-hydroxyketone rearrangement is potentially a conserved biosynthetic reaction in this molecular class. The biosynthetic alpha-hydroxyketone rearrangement was applied in a concise total synthesis of naphthomevalin, a prominent member of the napyradiomycin meroterpenes, and sheds further light on the mechanism of this unifying enzymatic transformation.

Zhang, JJ, Tang XY, Zhang M, Nguyen D, Moore BS.  2017.  Broad-host-range expression reveals native and host regulatory elements that influence heterologous antibiotic production in gram-negative bacteria. Mbio. 8   10.1128/mBio.01291-17   AbstractWebsite

Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-hostrange expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by similar to 60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility. IMPORTANCE Although Gram-positive heterologous hosts such as Streptomyces have been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs in Gram-negative proteobacterial expression hosts, and we leverage this platform to uncover regulatory elements that influence violacein expression from Pseudoalteromonas. Although it is often assumed that BGCs will be more successfully expressed in more closely related hosts, our work suggests that this may not be a general rule of thumb, as heterologous production of natural products can be influenced by specific host regulatory and/or biosynthetic elements, and the identity and effectiveness of those elements are difficult to predict. We argue for the use of a diverse set of heterologous hosts, which may also provide insights into BGC mechanism and function.