Diverse oxygenation patterns of natural products generated by secondary metabolic pathways in microorganisms and plants are largely achieved through the tailoring reactions catalysed by cytochrome P450 enzymes (P450s). P450s are a large family of oxidative hemoproteins found in all life forms from prokaryotes to humans. Understanding the reactivity and selectivity of these fascinating C-H bond-activating catalysts will advance their use in generating valuable pharmaceuticals and products for medicine, agriculture and industry. A major strength of this P450 group is its set of established enzyme-substrate relationships, the source of the most detailed knowledge on how P450 enzymes work. Engineering microbial-derived P450 enzymes to accommodate alternative substrates and add new functions continues to be an important near- and long-term practical goal driving the structural characterization of these molecules. Understanding the natural evolution of P450 structure-function should accelerate metabolic engineering and directed evolutionary approaches to enhance diversification of natural product structures and other biosynthetic applications.
In Nature, chiral natural products are usually produced in optically pure form; however, on occasion Nature is known to produce enantiomerically opposite metabolites. These enantiomeric natural products can arise in Nature from a single species, or from different genera and/or species. Extensive research has been carried out over the years in an attempt to understand the biogenesis of naturally occurring enantiomers, however, many fascinating puzzles and stereochemical anomalies still remain.
Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA suggesting a strong preference towards carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.
Polyketide synthase; germicidin; acyl carrier protein
In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia we chose to investigate the ET-743 (Yondelis®) biosynthetic pathway. This molecule is an approved anti-cancer agent obtained in low abundance (10−4–10−5% w/w) from the tunicate Ecteinascidia turbinata, and is generated in suitable quantities for clinical use by a lengthy semi-synthetic process. Based on structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anti-cancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogs through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.
Biosynthesis; ET-743; E. turbinata; metagenomics; metaproteomics; natural products; Pictet-Spenglerase; symbiont; tetrahydroisoquinoline; Yondelis
High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open information environment which enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up.
chemoinformatics; data analysis software; open source; high-throughput screening
Notoamides produced by Aspergillus spp. bearing the bicyclo [2.2.2] diazaoctane core structure with unusual structural diversity represent a compelling system to understand the biosynthesis of fungal prenylated indole alkaloids. Herein, we report the in vitro characterization of NotB, which catalyzes the indole 2,3-oxidation of notoamide E (13), leading to notoamide C (11) and D (12) through an apparent Pinacol-like rearrangement. This unique enzymatic reaction with high substrate specificity, together with the information derived from the precursor incorporation experiments using [13C]2-[15N]2 quadruply labeled notoamide S (10) demonstrates 10 as a pivotal branching point in notoamide biosynthesis.
Notoamide; fungi; prenylated indole alkaloids; biosynthesis; FAD monooxygenase
A strategy for regiochemical reversal of reductive macrocyclizations of aldehydes and terminal alkynes has been developed. Using an advanced synthetic intermediate directed towards the methymycin/neomethymycin class of macrolides, selective endocyclization provides the natural twelve-membered ring series, whereas ligand alteration enables selective exocyclization to provide access to the unnatural eleven-membered ring series. The twelve-membered ring adduct was converted to 10-deoxymethynolide, completing an efficient total synthesis of this natural product.
The cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by Micromonospora griseorubida). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in M. griseorubida. Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the mycCI disruption mutant; moreover, the mycCI gene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain M. griseorubida A11725. The mycG disruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The mycG gene was located immediately downstream of the self-resistance gene myrB. The mycG gene under the control of mycGp complemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the mycG gene under the control of myrBp was approximately 2-fold higher than that produced by the wild strain. In M. griseorubida, MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated.
The biosynthesis of fungal bicyclo[2.2.2]diazaoctane indole alkaloids with a wide spectrum of biological activities have attracted increasing interest. Their intriguing mode of assembly has long been proposed to feature a non-ribosomal peptide synthetase, a presumed intramolecular Diels-Alderase, a variant number of prenyltransferases, and a series of oxidases responsible for the diverse tailoring modifications of their cyclodipeptide-based structural core. Until recently, the details of these biosynthetic pathways have remained largely unknown due to lack of information on the fungal derived biosynthetic gene clusters. Herein, we report a comparative analysis of four natural product metabolic systems of a select group of bicyclo[2.2.2]diazaoctane indole alkaloids including (+)/(−)-notoamide, paraherquamide and malbrancheamide, in which we propose an enzyme for each step in the biosynthetic pathway based on deep annotation and on-going biochemical studies.
The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography.
Over eight different families of natural products, consisting of nearly seventy secondary metabolites, which contain the bicyclo[2.2.2]diazaoctane ring system, have been isolated from various Aspergillus, Penicillium, and Malbranchea species. Since 1968, these secondary metabolites have been the focus of numerous biogenetic, synthetic, taxonomic, and biological studies, and, as such, have made a lasting impact across multiple scientific disciplines. This review covers the isolation, biosynthesis, and biological activity of these unique secondary metabolites containing the bridging bicyclo[2.2.2]diazaoctane ring system. Furthermore, the diverse fungal origin of these natural products is closely examined and, in many cases, updated to reflect the currently accepted fungal taxonomy.
O-linked methylation of sugar substituents is a common modification in the biosynthesis of many natural products, and is catalyzed by multiple families of S-adenosyl-L-methioine (SAM or AdoMet) dependent methyltransferases. Mycinamicins, potent antibiotics from Micromonospora griseorubida, can be methylated at two positions on a 6-deoxyallose substituent. The first methylation is catalyzed by MycE, a SAM- and metal-dependent methyltransferase. Crystal structures were determined for MycE bound to the product S-adenosyl-L-homocysteine (SAH or AdoHcy) and magnesium, both with and without the natural substrate, mycinamicin VI. This represents the first structure of a natural product sugar methyltransferase in complex with its natural substrate. MycE is a tetramer of a two-domain polypeptide, comprising a C-terminal catalytic methyltransferase domain and an N-terminal auxiliary domain, which is important for quaternary assembly and for substrate binding. The symmetric MycE tetramer has a novel methyltransferase organization in which each of the four active sites is formed at the junction of three monomers within the tetramer. The active site structure supports a mechanism in which a conserved histidine acts as a general base, and the metal ion helps to position the methyl acceptor, and to stabilize a hydroxylate intermediate. A conserved tyrosine is suggested to support activity through interactions with the transferred methyl group from the SAM methyl donor. The structure of the free enzyme reveals a dramatic order-disorder transition in the active site relative to the SAH complexes, suggesting a mechanism for product/substrate exchange through concerted movement of five loops and the polypeptide C-terminus.
Polyketide natural products produced by type I modular polyketide synthases (PKSs) are key weapons in our drug arsenal. To reprogram these biosynthetic assembly lines we must first understand the steps that occur within the modular “black boxes”. Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site-occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain.
Cryptophycins are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogs with improved medicinal properties. Herein, we report a chemosynthetic route relying on the multifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy-acid moiety (unit D) into cryptophycin analogs. CrpD-M2 is a unique non-ribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic cryptophycin chain elongation intermediate analogs (SNAC-ABC) through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme bound seco-cryptophycin products were macrolactonized by the Crp thioesterase (TE). The analysis of these sequential steps was enabled through liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) analysis of enzyme bound intermediates and products. This novel chemoenzymatic synthesis of cryptophycin involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. This is the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new cryptophycin analogs.
The advanced natural product stephacidin A is proposed as a biosynthetic precursor to notoamide B in various Aspergillus species. Doubly 13C-labeled racemic stephacidin A was synthesized and fed to cultures of the terrestrial-derived fungus, Aspergillus versicolor NRRL 35600, and the marine-derived fungus, Aspergillus sp. MF297-2. Analysis of the metabolites revealed enantiospecific incorporation of intact (–)-stephacidin A into (+)-notoamide B in Aspergillus versicolor, and (+)-stephacidin A into (–)-notoamide B in Aspergillus sp. MF297-2. 13C-Labeled sclerotiamide was also isolated from both fungal cultures.
6-Hydroxydeoxybrevianamide E is proposed as a biosynthetic precursor to several advanced metabolites isolated from both marine-derived Aspergillus sp. and a terrestrial-derived Aspergillus versicolor. To verify the role of this reverse-prenylated indole alkaloid as an intermediate along the biosynthetic pathway, [13C]2-[15N]-6-hydroxydeoxybrevianamide E was synthesized and fed to Aspergillus versicolor. Analysis of the metabolites showed incorporation of the intermediate only into the natural product notoamide J.
Mitomycins are quinone-containing antibiotics, widely used as anti-tumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9β- and C9α-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR–S-adenosylhomocysteine (SAH) binary complex and MmcR–SAH-Mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 Å, respectively. The study revealed MmcR to adopt a common SAM-dependent O-MTase fold and the presence of a structurally-conserved active site general acid-base pair is consistent with a proton assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.
methyltransferase; natural product; biosynthesis; S-adenosyl-L-methionine; cancer; X-ray crystallography
Cryptophycins are potent anticancer agents isolated from Nostoc sp. ATCC 53789 and Nostoc sp. GSV 224. The most potent natural cryptophycin analogues retain a β-epoxide at the C2′-C3′ position of the molecule. A P450 epoxidase encoded by crpE recently identified from the cryptophycin gene cluster was shown to install this key functional group into cryptophycin-4 (Cr-4) to produce cryptophycin-2 (Cr-2) in a regio- and stereospecific manner. Here we report a detailed characterization of the CrpE epoxidase using an engineered maltose binding protein (MBP)-CrpE fusion. The substrate tolerance of the CrpE polypeptide was investigated with a series of structurally related cryptophycin analogues generated by chemoenzymatic synthesis. The enzyme specifically installed a β-epoxide between C2′ and C3′ of cyclic cryptophycin analogues. The kcat/Km values of the enzyme were determined to provide further insights into the P450 epoxidase catalytic efficiency affected by substrate structural variation. Finally, binding analysis revealed cooperativity of MBP-CrpE toward natural and unnatural desepoxy cryptophycin substrates.
Elucidation of natural product biosynthetic pathways provides important insights about the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here we show full reconstitution in vitro of an unusual multi-step oxidative cascade for post-assembly line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and an FAD-dependent oxidase (TamL), which act co-dependently through repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a heretofore unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C-10 oxidation of TirE.
Notoamide E, a short-lived secondary metabolite, has been proposed as a biosynthetic intermediate to several advanced metabolites isolated from Aspergillus versicolor. In order to verify the role of this indole alkaloid along the biosynthetic pathway, synthetic doubly 13C-labeled notoamide E was fed to Aspergillus versicolor. Analysis of the metabolites showed significant incorporation of notoamide E into the natural products notoamides C and D.
Aspergillus versicolor; biosynthesis; prenylated indole alkaloid
In vitro analysis of natural product biosynthetic gene products isolated from unculturable symbiotic bacteria is necessary to probe the functionalities of these enzymes. Herein, we report the biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) homolog implicated in β-branching at C13 and C21 of the core ring system from the bryostatin metabolic pathway (Bry). We confirmed the activity of BryR using two complementary methods, radio-SDS PAGE and Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry (FTICR-MS). The activity of BryR depended on pairing of the native acetoacetyl-BryM3 acceptor acyl carrier protein (ACP) with an appropriate donor acetyl-ACP from a heterologous HMGS cassette. Additionally, the ability of BryR to discriminate between various ACPs was assessed using a surface plasmon resonance (SPR)-based protein-protein binding assay. Our data suggest that specificity for a protein-bound acyl group is a distinguishing feature between HMGS homologs found in PKS or PKS/NRPS biosynthetic pathways and those of primary metabolism. These findings reveal an important example of molecular recognition between protein components that are essential for biosynthetic fidelity in natural product assembly and modification.
Proteus mirabilis causes complicated urinary tract infections (UTI). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly up-regulated genes (P ≤ 0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596–2605, encode putative siderophore systems. PMI0229-0239 encodes a nonribosomal peptide synthetase (NRPS)-independent siderophore (NIS) system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis, and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore reduce fitness in vivo. The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis.
Proteus; uropathogenesis; iron acquisition; ferri-siderophore
Stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a core bicyclo[2.2.2]diazaoctane ring system. These bioactive fungal secondary metabolites have a range of unusual structural and stereochemical features but their biosynthesis has remained uncharacterized. Herein, we report the first biosynthetic gene cluster for this class of fungal alkaloids based on whole genome sequencing of a marine-derived Aspergillus sp. Two central pathway enzymes catalyzing both normal and reverse prenyltransfer reactions were characterized in detail. Our results establish the early steps for creation of the prenylated indole alkaloid structure and suggest a scheme for the biosynthesis of stephacidin and notoamide metabolites. The work provides the first genetic and biochemical insights for understanding the structural diversity of this important family of fungal alkaloids.