The isolation, purification and complete characterization of the diterpene forskolin from nutritional supplements.

Forskolin (1) is a diterpene found in the Coleus forskohlii plant that has been examined for its medical properties resulting from adenylyl cyclase activation. This article describes a straightforward purification method of 1 from commercially available weight loss capsules. In addition, there has been some ambiguity with respect to the use of the name 'forskolin' to describe 1 and related diterpenes, which this report serves to eliminate. Herein we detail the complete spectroscopic characterization of purified 1 as well as its single crystal X-ray structure.

Phenylacetyl Coenzyme A, Not Phenylacetic Acid, Attenuates CepIR-Regulated Virulence in Burkholderia cenocepacia.

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

Lichen ketosynthase domains are not responsible for inoperative polyketide synthases in Ascomycota hosts.

Efforts by lichenologists to characterize lichen polyketide synthases (PKS) through heterologous expression experiments have so far proved unfruitful. A determination of systematic causes of failure is therefore required. Three hypotheses involving the ketosynthase (KS) domain of lichen polyketide synthases (PKS) from Cladonia uncialis are tested: (1) Horizontal versus vertical gene transfer; (2) Typical versus atypical active site residues; (3) Typical versus atypical tertiary protein structure and active site architecture. Phylogenetics, amino acid sequence alignment, and protein modelling indicate that C. uncialis PKS evolved through vertical transfer from Ascomycota fungi, possess Cys-His-His catalytic triads typical of KS from most organisms, and possess protein and catalytic site architecture identical to well-characterized KS from non-lichen organisms. Though the reason for lack of functional activity in heterologous hosts remains unknown, complications involving the KS are ruled out as a likely explanation. Heterologous translation of lichen PKS (or parts thereof) have not been reported. We demonstrate heterologous translation of two lichen KS domains in E. coli.

Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands.

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

Lichen Biosynthetic Gene Clusters. Part I. Genome Sequencing Reveals a Rich Biosynthetic Potential.

Lichens are symbionts of fungi and algae that produce diverse secondary metabolites with useful properties. Little is known of lichen natural product biosynthesis because of the challenges of working with lichenizing fungi. We describe the first attempt to comprehensively profile the genetic secondary metabolome of a lichenizing fungus. An Illumina platform combined with the Antibiotics and Secondary Metabolites Analysis Shell (FungiSMASH, version 4.0) was used to sequence and annotate assembled contigs of the fungal partner of Cladonia uncialis. Up to 48 putative gene clusters are described comprising type I and type III polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), hybrid PKS-NRPS, and terpene synthases. The number of gene clusters revealed by this work dwarfs the number of known secondary metabolites from C. uncialis, suggesting that lichenizing fungi have an unexplored biosynthetic potential.

Lichen Biosynthetic Gene Clusters Part II: Homology Mapping Suggests a Functional Diversity.

Lichens are renowned for their diverse natural products though little is known of the genetic programming dictating lichen natural product biosynthesis. We sequenced the genome of Cladonia uncialis and profiled its secondary metabolite biosynthetic gene clusters. Through a homology searching approach, we can now propose specific functions for gene products as well as the biosynthetic pathways that are encoded in several of these gene clusters. This analysis revealed that the lichen genome encodes the required enzymes for patulin and betaenones A-C biosynthesis, fungal toxins not known to be produced by lichens. Within several gene clusters, some (but not all) genes are genetically similar to genes devoted to secondary metabolite biosynthesis in Fungi. These lichen clusters also contain accessory tailoring genes without such genetic similarity, suggesting that the encoded tailoring enzymes perform distinct chemical transformations. We hypothesize that C. uncialis gene clusters have evolved by shuffling components of ancestral fungal clusters to create new series of chemical steps, leading to the production of hitherto undiscovered derivatives of fungal secondary metabolites.

A comprehensive catalogue of polyketide synthase gene clusters in lichenizing fungi.

Lichens are fungi that form symbiotic partnerships with algae. Although lichens produce diverse polyketides, difficulties in establishing and maintaining lichen cultures have prohibited detailed studies of their biosynthetic pathways. Creative, albeit non-definitive, methods have been developed to assign function to biosynthetic gene clusters in lieu of techniques such as gene knockout and heterologous expressions that are commonly applied to easily cultivatable organisms. We review a total of 81 completely sequenced polyketide synthase (PKS) genes from lichenizing fungi, comprising to our best efforts all complete and reported PKS genes in lichenizing fungi to date. This review provides an overview of the approaches used to locate and sequence PKS genes in lichen genomes, current approaches to assign function to lichen PKS gene clusters, and what polyketides are proposed to be biosynthesized by these PKS. We conclude with remarks on prospects for genomics-based natural products discovery in lichens. We hope that this review will serve as a guide to ongoing research efforts on polyketide biosynthesis in lichenizing fungi.

Synthetic cystic fibrosis sputum medium diminishes Burkholderia cenocepacia antifungal activity against Aspergillus fumigatus independently of phenylacetic acid production.

Phenylacetic acid (PAA), an intermediate of phenylalanine degradation, is emerging as a signal molecule in microbial interactions with the host. In this work, we explore the presence of phenylalanine and PAA catabolism in 3 microbial pathogens of the cystic fibrosis (CF) lung microbiome: Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aspergillus fumigatus. While in silico analysis of B. cenocepacia J2315 and A. fumigatus Af293 genome sequences showed complete pathways from phenylalanine to PAA, the P. aeruginosa PAO1 genome lacked several coding genes for phenylalanine and PAA catabolic enzymes. High-performance liquid chromatography analysis of supernatants from B. cenocepacia K56-2 detected PAA when grown in Luria-Bertani medium but not in synthetic cystic fibrosis sputum medium (SCFM). However, we were unable to identify PAA production by A. fumigatus or P. aeruginosa in any of the conditions tested. The inhibitory effect of B. cenocepacia on A. fumigatus growth was evaluated using agar plate interaction assays. Inhibition of fungal growth by B. cenocepacia was lessened in SCFM but this effect was not dependent on bacterial production of PAA. In summary, while we demonstrated PAA production by B. cenocepacia, we were not able to link this metabolite with the B. cenocepacia - A. fumigatus microbial interaction in CF nutritional conditions.

Identification of 6-Hydroxymellein Synthase and Accessory Genes in the Lichen Cladonia uncialis.

A transcribed polyketide synthase (PKS) gene has been identified in the lichen Cladonia uncialis. The complete nucleotide sequence of this PKS was determined from the amplified cDNA, and an assignment of individual domains was accomplished by homology searching using AntiSMASH. A scan of the complete genome sequence of C. uncialis revealed the accessory genes associated with this PKS gene. A homology search has identified that several genes in this cluster are similar to genes responsible for the biosynthesis of terrein in Aspergillus terreus. This permitted assignment of putative function to each of the genes in this new C. uncialis cluster. It is proposed that this gene cluster is responsible for the biosynthesis of a halogenated iscoumarin. This is the first report linking a gene cluster to a halogenated metabolite in lichen.

The attenuated virulence of a Burkholderia cenocepacia paaABCDE mutant is due to inhibition of quorum sensing by release of phenylacetic acid.

The phenylacetic acid degradation pathway of Burkholderia cenocepacia is active during cystic fibrosis-like conditions and is necessary for full pathogenicity of B. cenocepacia in nematode and rat infection models; however, the reasons for such requirements are unknown. Here, we show that the attenuated virulence of a phenylacetic acid catabolism mutant is due to quorum sensing inhibition. Unlike wild-type B. cenocepacia, a deletion mutant of the phenylacetyl-CoA monooxygenase complex (ΔpaaABCDE) released phenylacetic acid in the medium that favours infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the ΔpaaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild-type, due to phenylacetic acid consumption. In line with reduced detection of acyl-homoserine lactones in spent medium, the ΔpaaABCDE exhibited transcriptional inhibition of the quorum sensing system cepIR. Phenotypes repressed in ΔpaaABCDE, protease activity and pathogenicity against C. elegans, increased with exogenous N-octanoyl-L-homoserine lactone. Thus, we demonstrate that the attenuated phenotype of B. cenocepacia ΔpaaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N-octanoyl-L-homoserine lactone signalling. Further, we propose that active degradation of phenylacetic acid by B. cenocepacia during growth in cystic fibrosis-like conditions prevents accumulation of a quorum sensing inhibiting compound.

Limitations of the 'ambush hypothesis' at the single-gene scale: what codon biases are to blame?

Ribosomal frameshifting, a translational error, catastrophically alters the amino acid composition of the nascent protein by shifting the reading frame from the intended contiguous trinucleotide reading. Frameshift events waste energy and resources, and peptide products have unpredictable cytotoxic effects. The 'Ambush Hypothesis' (Seligmann and Pollock 2004, DNA Cell Biol 23:701-5) suggests there is a selective pressure favouring the evolution of out-of-frame ('hidden') stop codons. Although this hypothesis has gained empirical support through whole-genome studies, it is presently unknown whether it can be applied at a single-gene scale. Herein, we report such an investigation using the gene, polyketide synthase (PKS), among species of fungi. Contrary to expectation, genes presented with significantly lower number of hidden stop codons than expected in a selection-neutral model (p < 0.0005), suggesting both non-adherence to the ambush hypothesis as well as suppression of hidden stop codon evolution. It is known that there are multiple adaptive considerations determining codon selection during evolution, and that the information-holding potential of the genetic code is finite. We hypothesize that the reason for low hidden stops in PKS genes is due to competing 'codon biases' that are prioritized over the selective pressure favouring the emergence of hidden stops. Future studies of the ambush hypothesis in the context of other drivers of codon bias may allow this hypothesis to be molded into a comprehensive genetic theory that can be integrated within the broader genetic theory of codon bias and applied to the genetic code at any scale of analysis.

Isolation of Bioactive Metabolites from Soil Derived Fungus-Aspergillus fumigatus.

Fungi produce numerous secondary metabolites with intriguing biological properties for the health, industrial, and agricultural sectors. Herein, we report the high-yield isolation of phenolic natural products, N-formyl-4-hydroxyphenyl-acetamide 1 (~117 mg/L) and atraric acid 2 (~18 mg/L), from the ethyl acetate extract of the soil-derived fungus, Aspergillus fumigatus. The structures of compounds 1 and 2 were elucidated through the detailed spectroscopic analysis of NMR and LCMS data. These compounds were assayed for their antimicrobial activities. It was observed that compounds 1 and 2 exhibited strong inhibition against a series of fungal strains but only weak antibacterial properties against multi-drug-resistant strains. More significantly, this is the first known instance of the isolation of atraric acid 2 from a non-lichen fungal strain. We suggest the optimization of this fungal strain may exhibit elevated production of compounds 1 and 2, potentially rendering it a valuable source for the industrial-scale production of these natural antimicrobial compounds. Further investigation is necessary to establish the veracity of this hypothesis.

Crotonase catalysis enables flexible production of functionalized prolines and carbapenams.

The biocatalytic versatility of wildtype and engineered carboxymethylproline synthases (CMPSs) is demonstrated by the preparation of functionalized 5-carboxymethylproline derivatives methylated at C-2, C-3, C-4, or C-5 of the proline ring from appropriately substituted amino acid aldehydes and malonyl-coenzyme A. Notably, compounds with a quaternary center (at C-2 or C-5) were prepared in a stereoselective fashion by engineered CMPSs. The substituted-5-carboxymethyl-prolines were converted into the corresponding bicyclic ß-lactams using a carbapenam synthetase. The results demonstrate the utility of the crotonase superfamily enzymes for stereoselective biocatalysis, the amenability of carbapenem biosynthesis pathways to engineering for the production of new bicyclic ß-lactam derivatives, and the potential of engineered biocatalysts for the production of quaternary centers.

Unraveling usnic acid: a comparison of biosynthetic gene clusters between two reindeer lichen (Cladonia rangiferina and C. uncialis).

Lichenized fungi are known for their production of a diversity of secondary metabolites, many of which have broad biological and pharmacological applications. By far the most well-studied of these metabolites is usnic acid. While this metabolite has been well-known and researched for decades, the gene cluster responsible for its production was only recently identified from the species Cladonia uncialis. Usnic acid production varies considerably in the genus Cladonia, even among closely related taxa, and many species, such as C. rangiferina, have been inferred to be incapable of producing the metabolite based on analysis by thin-layer chromatography (TLC). We sequenced and examined the usnic acid biosynthetic gene clusters, or lack thereof, from four closely related Cladonia species (C. oricola, C. rangiferina, C. stygia, and C. subtenuis), and compare them against those of C. uncialis. We complement this comparison with tiered chemical profile analyses to confirm the presence or absence of usnic acid in select samples, using both HPLC and LC-MS. Despite long-standing reporting that C. rangiferina lacks the ability to produce usnic acid, we observed functional gene clusters from the species and detected usnic acid when extracts were examined by LC-MS. By contrast, C. stygia and C. oricola, have been previously described as lacking the ability to produce usnic acid, lacked the gene cluster entirely, and no usnic acid could be detected in C. oricola extracts via HPLC or LC-MS. This work suggests that chemical profiles attained through inexpensive and low-sensitivity methods like TLC may fail to detect low abundance metabolites that can be taxonomically informative. This study also bolsters understanding of the usnic acid gene cluster in lichens, revealing differences among domains of the polyketide synthase which may explain observed differences in expression. These results reinforce the need for comprehensive characterization of lichen secondary metabolite profiles with sensitive LC-MS methods.

Kinemage of action - proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level.

Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an external cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.

Pure rotational spectrum and ring inversion tunnelling of silacyclobutane.

The ground state pure rotational spectrum of silacyclobutane (SCB) (c-SiH(2)C(3)H(6)) has been investigated using both Fourier transform microwave (FTMW) and chirped pulse Fourier transform microwave (cp-FTMW) spectroscopies. Spectra of the (13)C, (29)Si, and (30)Si singly substituted isotopologues, in natural abundance, were recorded in the 6-24 GHz region along with those of the normal species. The ring inversion tunnelling splitting in the ground vibrational state was resolved and analyzed to determine the energy splitting of the two states: 75.7260(19) MHz. Structural analysis based on heavy atom substitution provided accurate geometric parameters including the bond lengths, bond angles, and ring puckering angle of the SCB ring backbone.

In situ imaging of usnic acid in selected Cladonia spp. by vibrational spectroscopy.

In this study, we demonstrate the first in situ detection of usnic acid (UA) in selected species of the lichen Cladonia, using FPA-FTIR imaging and Raman microscopy. Fruticose lichens present a variety of defensive mechanisms, one of which is the production of UA. This polyketide secondary metabolite, produced by certain lichenized fungi, has a protective function for the lichen that includes a strong absorption in the ultraviolet range. Upon confirming the distinct spectral signature of UA in lichen tissue, we mapped its distribution in Cladonia arbuscula, Cladonia uncialis and Cladonia sulphurina tissues. Spectroscopic images were obtained from cryosectioned lichen fragments embedded in media and from hand-sectioned fragments that were media-free. UA was present in the pycnidia, and younger walls of C. arbuscula and C. uncialis, the spore-producing region of a C. uncialis apothecium, and in both the younger and older soredia of C. sulphurina. The localization of UA in lichens is an important precursor to future work that includes the identification of the gene cluster responsible for its biosynthesis. Our results show that FTIR and Raman imaging can be an effective way to study the distribution of natural products in lichens with micron-scale precision.

Characterization of a Type-2 Diacylglycerol Acyltransferase from Haematococcus pluvialis Reveals Possible Allostery of the Recombinant Enzyme.

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies.

Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2.

Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein-protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.

Structural and mechanistic studies on N(2)-(2-carboxyethyl)arginine synthase.

N(2)-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the b-lactamase inhibitor clavulanic acid in Streptomyces clavuligerus. Crystal structures of tetrameric CEAS-ThDP in complex with the substrate analogues 5-guanidinovaleric acid (GVA) and tartrate, and a structure reflecting a possible enol(ate)-ThDP reaction intermediate are described. The structures suggest overlapping binding sites for the substrates D-glyceraldehyde-3-phosphate (D-G3P) and L-arginine, and are consistent with the proposed CEAS mechanism in which D-G3P binds at the active site and reacts to form an alpha,beta-unsaturated intermediate,which subsequently undergoes (1,4)-Michael addition with the alpha-amino group of L-arginine. Additional solution studies are presented which probe the amino acid substrate tolerance of CEAS, providing further insight into the L-arginine binding site. These findings may facilitate the engineering of CEAS towards the synthesis of alternative beta-amino acid products.