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.

The chemistry and biology of natural ribomimetics and related compounds.

Natural ribomimetics represent an important group of specialized metabolites with significant biological activities. Many of the activities, e.g., inhibition of seryl-tRNA synthetases, glycosidases, or ribosomes, are manifestations of their structural resemblance to ribose or related sugars, which play roles in the structural, physiological, and/or reproductive functions of living organisms. Recent studies on the biosynthesis and biological activities of some natural ribomimetics have expanded our understanding on how they are made in nature and why they have great potential as pharmaceutically relevant products. This review article highlights the discovery, biological activities, biosynthesis, and development of this intriguing class of natural products.

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4.

Cyanobacteria are prolific producers of natural products, and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters, and here we present the use of Anabaena sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native Anabaena 7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by coconjugation.

Premutilin Synthase: Ring Rearrangement by a Class II Diterpene Cyclase.

Biosynthesis of the complex diterpenoid antibiotic pleuromutilin relies on a bifunctional (di)terpene synthase, and here site-directed mutagenesis was used to knockout either of the two active sites. This enabled characterization of the novel ring contracted intermediate produced by the initiating class II diterpene cyclase active site. Quantum chemical calculations further indicate the importance of reactant configuration for this intriguing ring rearrangement.

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays.

The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344-in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.

Unexpected Biotransformation of the HDAC Inhibitor Vorinostat Yields Aniline-Containing Fungal Metabolites.

The diversity of genetically encoded small molecules produced by filamentous fungi remains largely unexplored, which makes these fungi an attractive source for the discovery of new compounds. However, accessing their full chemical repertoire under common laboratory culture conditions is a challenge. Epigenetic manipulation of gene expression has become a well-established tool for overcoming this obstacle. Here, we report that perturbation of the endophytic ascomycete Chalara sp. 6661, producer of the isofusidienol class of antibiotics, with the HDAC inhibitor vorinostat resulted in the production of four new modified xanthones. The structures of chalanilines A (1) and B (2) and adenosine-coupled xanthones A (3) and B (4) were determined by extensive NMR spectroscopic analyses, and the bioactivities of 1-4 were tested in antibiotic and cytotoxicity assays. Incorporation studies with deuterium-labeled vorinostat indicate that the aniline moiety in chalalanine A is derived from vorinostat itself. Our study shows that Chalara sp. is able to metabolize the HDAC inhibitor vorinostat to release aniline. This is a rare report of fungal biotransformation of the popular epigenetic modifier vorinostat into aniline-containing polyketides.

Identification of Elaiophylin Skeletal Variants from the Indonesian Streptomyces sp. ICBB 9297.

Four new elaiophylin macrolides (1-4), together with five known elaiophylins (5-9), have been isolated from cultures of the Indonesian soil bacterium Streptomyces sp. ICBB 9297. The new compounds have macrocyclic skeletons distinct from those of the known dimeric elaiophylins in that one or both of the polyketide chains contain(s) an additional pendant methyl group. Further investigations revealed that 1 and 2 were derived from 3 and 4, respectively, during isolation processes. Compounds 1-3 showed comparable antibacterial activity to elaiophylin against Staphylococcus aureus. However, interestingly, only compounds 1 and 3, which contain a pendant methyl group at C-2, showed activity against Mycobacterium smegmatis, whereas compound 2, which has two pendant methyl groups at C-2 and C-2', and the known elaiophylin analogues (5-7), which lack pendant methyl groups at C-2 and/or C-2', showed no activity. The production of 3 and 4 in strain ICBB 9297 indicates that one of the acyltransferase (AT) domains in the elaiophylin polyketide synthases (PKSs) can recruit both malonyl-CoA and methylmalonyl-CoA as substrates. Bioinformatic analysis of the AT domains of the elaiophylin PKSs revealed that the ela_AT7 domain contains atypical active site amino acid residues, distinct from those conserved in malonyl-CoA- or methylmalonyl-CoA-specific ATs.

Succinylated Apoptolidins from Amycolatopsis sp. ICBB 8242.

Two new apoptolidins, 2'-O-succinyl-apoptolidin A (11) and 3'-O-succinyl-apoptolidin A (12), were isolated from the culture broth of an Indonesian Amycolatopsis sp. ICBB 8242. These compounds inhibit the proliferation and viability of human H292 and HeLa cells. However, in contrast to apoptolidin A (1), they do not inhibit cellular respiration in H292 cells. It is proposed that apoptolidins are produced and secreted in their succinylated forms and 1 is the hydrolysis product of 11 and 12.

Depsipeptide companeramides from a Panamanian marine cyanobacterium associated with the coibamide producer.

Two new cyclic depsipeptides, companeramides A (1) and B (2), have been isolated from the phylogenetically characterized cyanobacterial collection that yielded the previously reported cancer cell toxin coibamide A (collected from Coiba Island, Panama). The planar structures of the companeramides, which contain 3-amino-2-methyl-7-octynoic acid (Amoya), hydroxy isovaleric acid (Hiva), and eight α-amino acid units, were established by NMR spectroscopy and mass spectrometry. The absolute configuration of each companeramide was assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of complete and partial hydrolysis products compared to commercial and synthesized standards. Companeramides A (1) and B (2) showed high nanomolar in vitro antiplasmodial activity but were not overtly cytotoxic to four human cancer cell lines at the doses tested.

Apoptolidins A and C activate AMPK in metabolically sensitive cell types and are mechanistically distinct from oligomycin A.

Apoptolidin A was first isolated as a secondary metabolite of a Nocardiopsis sp. and is the founding member of a family of potential selective cancer cell toxins. We now report the isolation, production and pharmacological characterization of apoptolidins A and C from an alternate actinomycete producer, an Amycolatopsis sp. from soil samples collected in Indonesia. We investigated the action of apoptolidins A and C in representative human glioblastoma cells, lung cancer cells and mouse embryonic fibroblasts (MEFs) to better understand the mechanism of action of the known apoptolidins. Shifts in cellular metabolism in intact cells and the status of the AMP-activated protein kinase (AMPK) stress pathway in response to apoptolidin A were entirely consistent with the actions of an ATP synthase inhibitor. We find the metabolic phenotype of the cell to be a critical determinant of apoptolidin sensitivity and the likely basis for cancer cell selectivity. The apoptolidins induce indirect activation of AMPK and trigger autophagy in sensitive cell types without significant inhibition of mTORC1. Human U87-MG glioblastoma cells and wild type MEFs showed increased phosphorylation of AMPK (Thr172), ACC (Ser79) and ULK1 (Ser555), whereas AMPKα-null MEFs and more glycolytic SF-295 glioblastoma cells lacked this response. Although both are reported to be selective inhibitors of mitochondrial ATP synthase, differences between apoptolidin- and oligomycin A-induced responses in cells indicate that the action of these macrolides is not identical.