Synthesis of the Carba-Analogs of the α-Pyranose and ß-Pyranose Forms of Sedoheptulose 7-Phosphate and Probing the Stereospecificity of Sedoheptulose 7-Phosphate Cyclases.

Sedoheptulose 7-phosphate (SH7P) cyclases are a subset of sugar phosphate cyclases that are known to catalyze the first committed step in many biosynthetic pathways in primary and secondary metabolism. Among them are 2-epi-5-epi-valiolone synthase (EEVS) and 2-epi-valiolone synthase (EVS), two closely related SH7P cyclases that catalyze the conversion of SH7P to 2-epi-5-epi-valiolone and 2-epi-valiolone, respectively. However, how these two homologous enzymes use a common substrate to produce stereochemically different products is unknown. Two competing hypotheses have been proposed for the stereospecificity of EEVS and EVS: (1) variation in aldol acceptor geometry during enzyme catalysis, and (2) preselection of the α-pyranose or ß-pyranose forms of the substrate by the enzymes. Yet, there is no direct evidence to support or rule out either of these hypotheses. Here we report the synthesis of the carba-analogs of the α-pyranose and ß-pyranose forms of SH7P and their use in probing the stereospecificity of ValA (EEVS from Streptomyces hygroscopicus subsp. jinggangensis) and Amir_2000 (EVS from Actinosynnema mirum DSM 43827). Kinetic studies of the enzymes in the presence of the synthetic compounds as well as docking studies of the enzymes with the α- and ß-pyranose forms of SH7P suggest that the inverted configuration of the products of EEVS and EVS is not due to the preselection of the different forms of the substrate by the enzymes.

Modifications of Protein-Bound Substrates by Trans-Acting Enzymes in Natural Products Biosynthesis.

Enzymatic modifications of small molecules are a common phenomenon in natural product biosynthesis, leading to the production of diverse bioactive compounds. In polyketide biosynthesis, modifications commonly take place after the completion of the polyketide backbone assembly by the polyketide synthases and the mature products are released from the acyl-carrier protein (ACP). However, exceptions to this rule appear to be widespread, as on-line hydroxylation, methyl transfer, and cyclization during polyketide assembly process are common, particularly in trans-AT PKS systems. Many of these modifications are catalyzed by specific domains within the modular PKS systems. However, several of the on-line modifications are catalyzed by stand-alone proteins. Those include the on-line Baeyer-Villiger oxidation, α-hydroxylation, halogenation, epoxidation, and methyl esterification during polyketide assembly, dehydrogenation of ACP-bound short fatty acids by acyl-CoA dehydrogenase-like enzymes, and glycosylation of ACP-bound intermediates by discrete glycosyltransferase enzymes. This review article highlights some of these trans-acting proteins that catalyze enzymatic modifications of ACP-bound small molecules in natural product biosynthesis.

Biological activities and metabolomic profiles of extracts from the marine sediment bacterium Nocardiopsis alba DP1B cultivated in different media.

The soil bacterium DP1B was isolated from a marine sediment collected off the coast of Randayan Island, Kalimantan Barat, Indonesia and identified based on 16S rDNA as Nocardiopsis alba. The bacterium was cultivated in seven different media (A1, ISP1, ISP2, ISP4, PDB, PC-1, and SCB) with three different solvents [distilled water, 5 % NaCl solution, artificial seawater (ASW)] combinations, shaken at 200 rpm, 30 °C, for 7 days. The culture broths were extracted with ethyl acetate and each extract was tested for its antimicrobial activity and brine shrimp lethality, and the chemical diversity was assessed using thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The result showed that almost all extracts showed antibacterial but not antifungal activity, whereas their brine shrimp toxicity levels vary from high to low. The best medium/solvent combinations for antibacterial activity and toxicity were PC-1 (in either distilled water, 5% NaCl solution, or ASW) and SCB in ASW. Different chemical diversity profiles were observed on TLC, GC-MS, and LC-MS/MS. Extracts from the PC-1 cultures seem to contain a significant number of cyclic dipeptides, whereas those from the SCB cultures contain sesquiterpenes, indicating that media and solvent compositions can affect the secondary metabolite profiles of DP1B. In addition, untargeted metabolomic analyses using LC-MS/MS showed many molecular ions that did not match with those in the Global Natural Products Social Molecular Networking (GNPS) database, suggesting that DP1B has great potential as a source of new natural products.

Modifications of Acyl Carrier Protein-Bound Glycosylated Polyketides in Pactamycin Biosynthesis.

The potent antitumor antibiotic pactamycin is an aminocyclopentitol-containing natural product produced by the soil bacterium Streptomyces pactum. Recent studies showed that the aminocyclopentitol unit is derived from N-acetyl-D-glucosamine, which is attached to an acyl carrier protein (ACP)-bound polyketide by a glycosyltransferase enzyme, PtmJ. Here, we report a series of post-glycosylation modifications of the sugar moiety of the glycosylated polyketide while it is still attached to the carrier protein. In vitro reconstitution of PtmS (an AMP-ligase), PtmI (an ACP), PtmJ, PtmN (an oxidoreductase), PtmA (an aminotransferase), and PtmB (a putative carbamoyltransferase) showed that the N-acetyl-D-glucosamine moiety of the glycosylated polyketide is first oxidized by PtmN and then transaminated by PtmA to give ACP-bound 3-amino-3-deoxy-N-acetyl-D-glucosaminyl polyketide. The amino group is then coupled with carbamoyl phosphate by PtmB to give a urea functionality. We also show that PtmG is a deacetylase that hydrolyses the C-2 N-acetyl group to give a free amine.

Genome features and secondary metabolite potential of the marine symbiont Streptomyces sp. RS2.

Streptomyces sp. RS2 was isolated from an unidentified sponge collected around Randayan Island, Indonesia. The genome of Streptomyces sp. RS2 consists of a linear chromosome of 9,391,717 base pairs with 71.9% of G + C content, 8270 protein-coding genes, as well as 18 rRNA and 85 tRNA loci. Twenty-eight putative secondary metabolites biosynthetic gene clusters (BGCs) were identified in the genome sequence. Nine of them have 100% similarity to BGCs for albaflavenone, α-lipomycin, coelibactin, coelichelin, ectoine, geosmin, germicidin, hopene, and lanthionine (SapB). The remaining 19 BGCs have low (< 50%) or moderate (50-80%) similarity to other known secondary metabolite BGCs. Biological activity assays of extracts from 21 different cultures of the RS2 strain showed that SCB ASW was the best medium for the production of antimicrobial and cytotoxic compounds. Streptomyces sp. RS2 has great potential to be a producer of novel secondary metabolites, particularly those with antimicrobial and antitumor activities.

Synthesis and Anti-Inflammatory Activity of the Natural Cyclooxygenase-2 Inhibitor Axinelline A and Its Analogues.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used medications to treat conditions such as arthritis, pain, and fever. They reduce inflammation by inhibiting cyclooxygenase (COX) enzymes that catalyze the committed step in prostaglandin (PG) biosynthesis. Despite their significant therapeutic benefits, many NSAIDS have undesirable adverse effects. The aim of this study was to discover novel COX inhibitors from natural sources. Here, we describe the synthesis and anti-inflammatory activity of the COX-2 inhibitor axinelline A (A1), which was isolated from Streptomyces axinellae SCSIO02208, and its analogues. Compared to the synthetic analogues, the natural product A1 has stronger COX inhibitory activity. Although A1 is more active against COX-2 than COX-1, its selectivity index is low; therefore, it may be classified as a nonselective COX inhibitor. Its overall activity is comparable to the clinically used drug diclofenac. In silico studies showed that A1 binds to COX-2 in a similar manner to diclofenac. Inhibition of COX enzymes by A1 in LPS-stimulated murine RAW264.7 macrophages resulted in suppression of the NF-κB signaling pathway, leading to reduced expression of pro-inflammatory factors such as iNOS, COX-2, TNF-α, IL-6, and IL-1ß and reduced production of PGE2, NO, and ROS. The potent in vitro anti-inflammatory activity of A1, together with its lack of cytotoxicity, makes it an attractive candidate for a new anti-inflammatory lead.

Biochemical Characterization of GacI, a Bifunctional Glycosyltransferase-Phosphatase Enzyme Involved in Acarbose Biosynthesis in Streptomyces glaucescens GLA.O.

Acarbose, a pseudotetrasaccharide produced by several strains of Actinoplanes and Streptomyces, is an α-glucosidase inhibitor clinically used to control type II diabetes. Bioinformatic analysis of the biosynthetic gene clusters of acarbose in Actinoplanes sp. SE50/110 (the acb cluster) and Streptomyces glaucescens GLA.O (the gac cluster) revealed their distinct genetic organizations and presumably biosynthetic pathways. However, to date, only the acarbose pathway in the SE50/110 strain has been extensively studied. Here, we report that GacI, one of the proteins that appear to be different between the two pathways, is a bifunctional glycosyltransferase family 5 (GT5)-phosphatase (PP) enzyme that functions at two different steps in acarbose biosynthesis in S. glaucescens GLA.O. In the acb pathway, the GT and the PP reactions are performed by two different enzymes. Truncated GacI proteins having only the GT or the PP domain showed comparable catalytic activity with the full-length GacI, indicating that domain separation does not significantly affect their respective catalytic activity. GacI, which is widely distributed in many Streptomyces, represents the first example of naturally occurring GT5-PP bifunctional enzymes biochemically characterized.

EDB Gene Cluster-Dependent Indole Production Is Responsible for the Ability of Pseudomonas fluorescens NZI7 to Repel Grazing by Caenorhabditis elegans.

The "EDB" (from "edible") gene cluster, a variant of the ebo cluster of genes found in many bacteria and algae, allows Pseudomonas fluorescens NZI7 (referred to here as "NZI7") to repel grazing by the nematode Caenorhabditis elegans. The mechanism underlying this phenotype is unknown. Here we report that the EDB cluster is involved in the conversion of tryptophan to (1H-indol-3-yl)-oxoacetamide, indole 3-aldehyde, and other indole-derived compounds. Inactivation of the EDB genes in NZI7 resulted in mutants that lack the ability to excrete indole-derived compounds as well as the ability to repel C. elegans. Heterologous expression of the NZI7 EDB cluster in E. coli cultivated in minimal M9 medium containing 2 mM l-tryptophan also released indole derivatives including tryptophol, 3-(hydroxyacetyl)indole, colletotryptin E, and two new dimeric indoles. Expression of the NZI7 EDB cluster in E. coli, cultured in minimal M9 medium and lacking tryptophan, did not produce detectable levels of indole derivatives. Both (1H-indol-3-yl)-oxoacetamide and indole 3-aldehyde showed repellent activity against C. elegans, revealing the mechanism underlying the ability of P. fluorescens NZI7 to repel grazing by C. elegans.

Exophilone, a Tetrahydrocarbazol-1-one Analogue with Anti-Pulmonary Fibrosis Activity from the Deep-Sea Fungus Exophiala oligosperma MCCC 3A01264.

A new compound, exophilone (1), together with nine known compounds (2-10), were isolated from a deep-sea-derived fungus, Exophiala oligosperma. Their chemical structures, including the absolute configuration of 1, were elucidated using nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and electronic circular dichroism (ECD) calculation. Compounds were preliminarily screened for their ability to inhibit collagen accumulation. Compounds 1, 4, and 7 showed weaker inhibition of TGF-ß1-induced total collagen accumulation in compared with pirfenidone (73.14% inhibition rate). However, pirfenidone exhibited cytotoxicity (77.57% survival rate), while compounds 1, 4, and 7 showed low cytotoxicity against the HFL1 cell line. Particularly, exophilone (1) showed moderate collagen deposition inhibition effect (60.44% inhibition rate) and low toxicity in HFL1 cells (98.14% survival rate) at a concentration of 10 µM. A molecular docking study suggests that exophilone (1) binds to both TGF-ß1 and its receptor through hydrogen bonding interactions. Thus, exophilone (1) was identified as a promising anti-pulmonary fibrosis agent. It has the potential to be developed as a drug candidate for pulmonary fibrosis.

Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis.

Glycosylation is a common modification reaction in natural product biosynthesis and has been known to be a post-assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis, glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation and in vitro pathway reconstitution, we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-(3-aminophenyl)3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free ß-ketoacid that subsequently undergoes decarboxylation.

Identification and Biological Activity of NFAT-133 Congeners from Streptomyces pactum.

The soil bacterium Streptomyces pactum ATCC 27456 produces a number of polyketide natural products. Among them is NFAT-133, an inhibitor of the nuclear factor of activated T cells (NFAT) that suppresses interleukin-2 (IL-2) expression and T cell proliferation. Biosynthetic gene inactivation in the ATCC 27456 strain revealed the ability of this strain to produce other polyketide compounds including analogues of NFAT-133. Consequently, seven new derivatives of NFAT-133, TM-129-TM-135, together with a known compound, panowamycin A, were isolated from the culture broth of S. pactum ATCC 27456 ΔptmTDQ. Their chemical structures were elucidated on the basis of their HRESIMS, 1D and 2D NMR spectroscopy, and ECD calculation and spectral data. NFAT-133, TM-132, TM-135, and panowamycin A showed no antibacterial activity or cytotoxicity, but weakly reduced the production of LPS-induced nitric oxide in RAW264.7 cells in a dose-dependent manner. A revised chemical structure of panowamycin A and proposed modes of formation of the new NFAT-133 analogues are also presented.

Biotransformations of anthranilic acid and phthalimide to potent antihyperlipidemic alkaloids by the marine-derived fungus Scedosporium apiospermum F41-1.

A new diphenylamine derivative, scediphenylamine A (1), together with six phthalimide derivatives (2-7) and ten other known compounds (8-17) were obtained from the marine-derived fungus Scedosporium apiospermum F41-1 fed with synthetically prepared anthranilic acid and phthalimide. The structure and absolute configuration of the new compound were determined by HRMS, NMR, and X-ray crystallography. Evaluation of their lipid-lowering effect in 3T3-L1 adipocytes showed that scediphenylamine A (1), N-phthaloyl-tryptophan-methyl ester (4), 5-(1,3-dioxoisoindolin-2-yl) pentanamide (5), perlolyrine (10) and flazine (11) significantly reduced triglyceride level in 3T3-L1 cells by inhibiting adipogenic differentiation and synthesis with the EC50 values of 4.39, 2.79, 3.76, 0.09, and 4.52 µM, respectively. Among them, perlolyrine (10) showed the most potent activity, making it a candidate for further development as a potential agent to treat hyperlipidemia.

Phomaligols F-I, polyoxygenated cyclohexenone derivatives from marine-derived fungus Aspergillus flavus BB1.

By tracing the 13C NMR resonances for carbonyls and enols, four new oxidized phomaligol derivatives, phomaligols F-I (1-4), along with seven known compounds (5-11) were isolated from the culture of the fungus Aspergillus flavus BB1 isolated from the marine shellfish Meretrix meretrix collected on Hailing Island, Yangjiang, China. The chemical structures and the absolute configurations of the new compounds were elucidated by MS, NMR, ECD, optical rotation, and 13C NMR calculations. Compounds 1 and 2 represent the first examples of phomaligol derivatives that contain an unusual bicyclic skeleton. All isolated compounds were tested for their cytotoxic activity. Among them, sporogen-AO 1 (8) showed potent inhibitory activity against the cancer cell lines A549, H1299, SK-BR-3, and HCT116 with IC50 values of 0.13, 0.78, 1.19, and 1.32 µM, respectively. Phomaligol G (2) displayed cytotoxic activity against the A549 and H1299 cell lines with IC50 values of 46.86 and 51.87 µM respectively. Additionally, phomaligol H (3) demonstrated cytotoxic activity against the A549 cell line with an IC50 value of 65.53 µM. Mechanistic studies of compound 8 showed that it induced apoptosis of HCT116 cells in a dose-dependent manner.

Potential Antidiabetic Fumiquinazoline Alkaloids from the Marine-Derived Fungus Scedosporium apiospermum F41-1.

Fumiquinazoline alkaloids have attracted much attention from medicinal and natural product chemists due to their interesting structures and biological potential. In this study, three new and 12 known fumiquinazoline alkaloids were isolated and characterized from the marine fungus Scedosporium apiospermum F41-1. The structures of the new compounds and their absolute configurations were determined using NMR spectroscopy, ECD, and OR calculations. The compounds were evaluated for their antidiabetic potential by determining their triglyceride-promoting activity using 3T3-L1 adipocytes. One of the new compounds, scequinadoline J (14), as well as scequinadolines D (9) and E (10), was found to promote triglyceride accumulation in 3T3-L1 cells. Scequinadoline D (9) demonstrated the most potent activity, with an EC50 value of 0.27 ± 0.03 µM. Quantitative polymerase chain reaction experiments suggested that scequinadoline D (9) acts through activation of the PPARγ pathway. It stimulated the mRNA expression of PPARγ, AMPKα, C/EBPα, LXRα, SCD-1, and FABP4. In addition, its triglyceride-promoting efficacy could be blocked by a double dose of the PPARγ antagonist GW9662. These results indicated that scequinadoline D (9) is a potent insulin sensitizer that targets adipocytes and may be useful for the treatment of type 2 diabetes mellitus after further investigation.

l-Tryptophan Induces a Marine-Derived Fusarium sp. to Produce Indole Alkaloids with Activity against the Zika Virus.

The effects of l-tryptophan supplementation on secondary metabolite production in the marine-derived fungus Fusarium sp. L1 were investigated by culturing the fungus in GPY medium with and without the amino acid. HPLC analysis of the products showed distinct metabolite profiles between the two cultures. The 1H NMR spectrum of the EtOAc extract of the culture supplemented with l-tryptophan displayed a series of characteristic aromatic proton signals (δH 6.50-8.50) and NH signals (δH 10.50-11.50) that were not observed in those from cultures not supplemented with l-tryptophan. Subsequently, 23 distinct indole alkaloids, including six new compounds, fusaindoterpenes A and B (1 and 2), fusariumindoles A-C (3-5), and (±)-isoalternatine A (6), together with 17 known compounds, were obtained from this culture. Fusaindoterpene A (1) contains a 6/9/6/6/5 heterocyclic system. Their chemical structures were determined by analysis of HRMS, NMR spectroscopy, optical rotation calculation, ECD calculation, and single-crystal X-ray diffraction data. Compounds 2, 9, and 15 displayed inhibitory activity against the Zika virus (ZIKV) in a standard plaque assay with EC50 values of 7.5, 4.2, and 5.0 µM, respectively, while not showing significant cell cytotoxicity against the A549 adenocarcinomic human alveolar basal epithelial cell line.

l-Phenylalanine Alters the Privileged Secondary Metabolite Production in the Marine-Derived Fungus Trichoderma erinaceum F1-1.

The effects of a single-amino-acid culture strategy on secondary metabolite production in the marine-derived fungus Trichoderma erinaceum F1-1 were investigated by culturing the fungus in GPY medium supplemented or not supplemented with l-phenylalanine. A suite of secondary metabolites, including seven terpenoids (1-7) and one polyketide (8), among which are four new compounds, harziandione A (1), cyclonerodiols A and B (3, 4), and trichodermaerin A (6), were isolated from the GPY medium without l-phenylanine, whereas 18 aromatic compounds (9-26), including six new compounds, trichoderolides A-F (9, 10, and 14-17), were isolated from the culture grown in the GPY medium with l-phenylalanine. The structures of the new compounds were determined by high-resolution mass spectrometry, NMR spectroscopic analysis, optical rotation calculations, chemical methods, and X-ray crystallography. Compounds 10, 12, 13, and 26 exhibited cytotoxic activities against MDA-MB-435 human melanocyte cancer cells. Compound 26 was cytotoxic to A549 adenocarcinomic human alveolar basal epithelial cells.

Armeniaspirol Antibiotic Biosynthesis: Chlorination and Oxidative Dechlorination Steps Affording Spiro[4.4]non-8-ene.

Armeniaspirols are potent antibiotics containing an unusual spiro[4.4]non-8-ene moiety. Herein, we describe the cloning and functional analysis of the armeniaspirol biosynthetic gene cluster. Gene-inactivation studies and subsequent isolation of previously unknown biosynthetic intermediates shed light on intriguing biosynthetic details. Remarkably, deletion of ams15, which encodes a protein bearing a flavin-binding domain, led to the accumulation of several non-spiro intermediates with various numbers of chlorine substitutions on the pyrrole moiety. The di- and trichloropyrrole species were converted by Streptomyces albus expressing Ams15 into mono- and dichlorinated spiro derivatives, respectively. In addition, in vitro conversion of these non-spiro intermediates into des-N-methyl spiro intermediates by the cell lysate of the same recombinant strain proved Ams15 to be responsible for spiro formation through oxidative dehalogenation.

The secondary metabolite pactamycin with potential for pharmaceutical applications: biosynthesis and regulation.

The antitumor antibiotic pactamycin is a highly substituted aminocyclopentitol-derived secondary metabolite produced by the soil bacterium Streptomyces pactum. It has exhibited potent antibacterial, antitumor, antiviral, and antiprotozoal activities. Despite its outstanding biological activities, the complex chemical structure and broad-spectrum toxicity have hampered its development as a therapeutic, limiting its contribution to biomedical science to a role as a molecular probe for ribosomal function. However, a detailed understanding of its biosynthesis and how the biosynthesis is regulated has made it possible to tactically design and produce new pactamycin analogues, some of which have shown improved pharmacological properties. This mini-review describes the biosynthesis, regulation, engineered production, and biological activities of pactamycin and its congeners. It also highlights the suitability of biosynthetic methods as a feasible approach to generate new analogues of complex natural products and underscores the importance of utilizing biosynthetic enzymes as tools for chemoenzymatic production of structurally diverse bioactive compounds.

Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum.

Pactamycin, a structurally unique aminocyclitol natural product isolated from Streptomyces pactum, has potent antibacterial, antitumor, and anti-protozoa activities. However, its production yields under currently used culture conditions are generally low. To understand how pactamycin biosynthesis is regulated and explore the possibility of improving pactamycin production in S. pactum, we investigated the transcription regulations of pactamycin biosynthesis. In vivo inactivation of two putative pathway-specific regulatory genes, ptmE and ptmF, resulted in mutant strains that are not able to produce pactamycin. Genetic complementation using a cassette containing ptmE and ptmF integrated into the S. pactum chromosome rescued the production of pactamycin. Transcriptional analysis of the ΔptmE and ΔptmF strains suggests that both genes control the expression of the whole pactamycin biosynthetic gene cluster. However, attempts to overexpress these regulatory genes by introducing a second copy of the genes in S. pactum did not improve the production yield of pactamycin. We discovered that pactamycin biosynthesis is sensitive to phosphate regulation. Concentration of inorganic phosphate higher than 2 mM abolished both the transcription of the biosynthetic genes and the production of the antibiotic. Draft genome sequencing of S. pactum and bioinformatics studies revealed the existence of global regulatory genes, e.g., genes that encode a two-component PhoR-PhoP system, which are commonly involved in secondary metabolism. Inactivation of phoP did not show any significant effect to pactamycin production. However, in the phoP::aac(3)IV mutant, pactamycin biosynthesis is not affected by external inorganic phosphate concentration.

The sedoheptulose 7-phosphate cyclases and their emerging roles in biology and ecology.

Covering up to: 1999-2016This highlight covers a family of enzymes of growing importance, the sedoheptulose 7-phosphate cyclases, initially of interest due to their involvement in the biosynthesis of pharmaceutically relevant secondary metabolites. More recently, these enzymes have been found throughout Prokarya and Eukarya, suggesting their broad potential biological roles in nature.