Final piece to the Fusarium pigmentation puzzle - Unraveling of the phenalenone biosynthetic pathway responsible for perithecial pigmentation in the Fusarium solani species complex.

The Fusarium solani species complex (FSSC) is comprised of important pathogens of plants and humans. A distinctive feature of FSSC species is perithecial pigmentation. While the dark perithecial pigments of other Fusarium species are derived from fusarubins synthesized by polyketide synthase 3 (PKS3), the perithecial pigments of FSSC are derived from an unknown metabolite synthesized by PKS35. Here, we confirm in FSSC species Fusarium vanettenii that PKS35 (fsnI) is required for perithecial pigment synthesis by deletion analysis and that fsnI is closely related to phnA from Penicillium herquei, as well as duxI from Talaromyces stipentatus, which produce prephenalenone as an early intermediate in herqueinone and duclauxin synthesis respectively. The production of prephenalenone by expression of fsnI in Saccharomyces cerevisiae indicates that it is also an early intermediate in perithecial pigment synthesis. We next identified a conserved cluster of 10 genes flanking fsnI in F. vanettenii that when expressed in F. graminearum led to the production of a novel corymbiferan lactone F as a likely end product of the phenalenone biosynthetic pathway in FSSC.

Proteomic Changes in Methicillin-Resistant Staphylococcus aureus Exposed to Cannabinoids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to ß-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In this study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

Mechanistic basis of substrate-O2 coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study.

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x2-y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase.

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.

Upgrading the Nutritional Value of PKC Using a Bacillus subtilis Derived Monocomponent ß-Mannanase.

Palm kernel cake (PKC) is an abundant side stream that can only be added to non-ruminant feed in small concentrations due to its content of antinutritional factors, mainly galactomannan, which cannot be digested by non-ruminants. ß-mannanases can be added to partially hydrolyze galactomannan to form mannose oligosaccharides, which are known to be prebiotic. We here investigate the action of a ß-mannanase from B. subtilis on PKC by colorimetry, NMR and fluorescence microscopy. The amount of mannan oligosaccharides in solution was significantly increased by the ß-mannanase and their degree of polymerization (DP) was significantly reduced. There was a dose-response behavior in that larger ß-mannanase concentrations increased the amount of soluble mannose oligosaccharides while reducing their average DP. Using confocal immunofluorescence microscopy, solubilization of galactomannan in PKC was clearly visualized. Images show a clear disruption of the cellulose and galactomannan structures of the PKC cell walls. We thus show in this study that using commercial dosages of ß-mannanase on PKC can lead to formation of prebiotic compounds. Thus, this study suggests that utilization of PKC in poultry feed formulation might be increased by addition of a ß-mannanase and would improve the return on investment.

Metabolic changes during carbon monoxide poisoning: An experimental study.

Carbon monoxide (CO) is the leading cause of death by poisoning worldwide. The aim was to explore the effects of mild and severe poisoning on blood gas parameters and metabolites. Eleven pigs were exposed to CO intoxication and had blood collected before and during poisoning. Mild CO poisoning (carboxyhaemoglobin, COHb 35.2 ± 7.9%) was achieved at 32 ± 13 minutes, and severe poisoning (69.3 ± 10.2% COHb) at 64 ± 23 minutes from baseline (2.9 ± 0.5% COHb). Blood gas parameters and metabolites were measured on a blood gas analyser and nuclear magnetic resonance spectrometer, respectively. Unsupervised principal component, analysis of variance and Pearson's correlation tests were applied. A P-value ≤ .05 was considered statistically significant. Mild poisoning resulted in a 28.4% drop in oxyhaemoglobin (OHb) and 12-fold increase in COHb, while severe poisoning in a 65% drop in OHb and 24-fold increase in COHb. Among others, metabolites implicated in regulation of metabolic acidosis (lactate, P < .0001), energy balance (pyruvate, P < .0001; 3-hydroxybutyrc acid, P = .01), respiration (citrate, P = .007; succinate, P = .0003; fumarate, P < .0001), lipid metabolism (glycerol, P = .002; choline, P = .0002) and antioxidant-oxidant balance (glutathione, P = .03; hypoxanthine, P < .0001) were altered, especially during severe poisoning. Our study adds new insights into the deranged metabolism of CO poisoning and leads the way for further investigation.

The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor.

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia-causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84-2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.

Metabolic fingerprint of progression of chronic hepatitis B: changes in the metabolome and novel diagnostic possibilities.

INTRODUCTION: Chronic hepatitis B (CHB) affects 257 million individuals worldwide with an annual estimated mortality rate of 880,000 individuals. Accurate diagnosis of the stage of disease is difficult, and there is considerable uncertainty concerning the optimal point in time, when treatment should be started. OBJECTIVES: By analyzing and comparing the metabolomes of patients at different stages of CHB and comparing them to healthy individuals, we want to determine the metabolic signature of disease progression and develop a more accurate metabolome-based method for diagnosis of disease progression ultimately giving a better basis for treatment decisions. METHODS: In this study, we used the combination of transient elastography and serum metabolomics of 307 serum samples from a group of 90 patients with CHB before and under treatment (with a follow-up time up to 10 years) at different progression stages over the clinical phases and 43 healthy controls.. RESULTS: Our data show that the metabolomics approach can successfully discover CHB changing from the immune tolerance to the immune clearance phase and show distinctive metabolomes from different medical treatment stages. Perturbations in ammonia detoxification, glutamine and glutamate metabolism, methionine metabolism, dysregulation of branched-chain amino acids, and the tricarboxylic acid (TCA) cycle are the main factors involved in the progression of the disease. Fluctuations increasing in aspartate, glutamate, glutamine, methionine and 13 other metabolites are fingerprints of progression. CONCLUSIONS: The metabolomics approach may expand the diagnostic armamentarium for patients with CHB. This method can provide a more detailed decision basis for starting medical treatment.

Cyclic, Hydrophobic Hexapeptide Fusahexin Is the Product of a Nonribosomal Peptide Synthetase in Fusarium graminearum.

The plant pathogenic fungus Fusarium graminearum is known to produce a wide array of secondary metabolites during plant infection. This includes several nonribosomal peptides. Recently, the fusaoctaxin (NRPS5/9) and gramilin (NRPS8) gene clusters were shown to be induced by host interactions. To widen our understanding of this important pathogen, we investigated the involvement of the NRPS4 gene cluster during infection and oxidative and osmotic stress. Overexpression of NRPS4 led to the discovery of a new cyclic hexapeptide, fusahexin (1), with the amino acid sequence cyclo-(d-Ala-l-Leu-d-allo-Thr-l-Pro-d-Leu-l-Leu). The structural analyses revealed an unusual ether bond between a proline Cδ to Cß of the preceding threonine resulting in an oxazine ring system. The comparative genomic analyses showed that the small gene cluster only encodes an ABC transporter in addition to the five-module nonribosomal peptide synthetase (NRPS). Based on the structure of fusahexin and the domain architecture of NRPS4, we propose a biosynthetic model in which the terminal module is used to incorporate two leucine units. So far, iterative use of NRPS modules has primarily been described for siderophore synthetases, which makes NRPS4 a rare example of a fungal nonsiderophore NRPS with distinct iterative module usage.

Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper (Bothrops atrox).

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead (Bothrops atrox). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.

Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel.

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in the CALM genes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the CaV1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca2+ by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca2+-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca2+-dependent inactivation of the voltage-gated calcium channel.

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca2+ binding and cause misfolding.

KEY POINTS: Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage-gated calcium channel CaV 1.2. Arrhythmia mutations in CaM can lead to loss of Ca2+ binding, uncoupling of Ca2+ binding cooperativity, misfolding of the EF-hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease. ABSTRACT: Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage-gated Ca2+ channel CaV 1.2 and the ryanodine receptor (RyR2), in a mutation-specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C-terminal lobe mutants and an N-terminal lobe mutant in complex with the CaV 1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF-hands within the C-lobe and loss of Ca2+ binding in EF-hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca2+ -saturating conditions when unbound to the IQ domain. Ca2+ binding to a pair of EF-hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca2+ ions bound to the C-lobe, possibly disrupting the cooperativity. An N-lobe variant (N54I), which causes severe stress-induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of CaV 1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease.

Heterologous Expression of the Core Genes in the Complex Fusarubin Gene Cluster of Fusarium Solani.

Through stepwise recreation of the biosynthetic gene cluster containing PKS3 from Fusarium solani, it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in Saccharomyces cerevisiae. This was achieved through sequential transformation associated recombination (TAR) cloning of FvPPT, fsr1, fsr2, and fsr3 into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in S. cerevisiae. The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1-4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L.

19F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by 19F NMR.

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum.

Filamentous fungi such as species from the genus Fusarium are capable of producing a wide palette of interesting metabolites relevant to health, agriculture and biotechnology. Secondary metabolites are formed from large synthase/synthetase enzymes often encoded in gene clusters containing additional enzymes cooperating in the metabolite's biosynthesis. The true potential of fungal metabolomes remain untapped as the majority of secondary metabolite gene clusters are silent under standard laboratory growth conditions. One way to achieve expression of biosynthetic pathways is to clone the responsible genes and express them in a well-suited heterologous host, which poses a challenge since Fusarium polyketide synthase and non-ribosomal peptide synthetase gene clusters can be large (e.g. as large as 80 kb) and comprise several genes necessary for product formation. The major challenge associated with heterologous expression of fungal biosynthesis pathways is thus handling and cloning large DNA sequences. In this paper we present the successful workflow for cloning, reconstruction and heterologous production of two previously characterized Fusarium pseudograminearum natural product pathways in Fusarium graminearum. In vivo yeast recombination enabled rapid assembly of the W493 (NRPS32-PKS40) and the Fusarium Cytokinin gene clusters. F. graminearum transformants were obtained through protoplast-mediated and Agrobacterium tumefaciens-mediated transformation. Whole genome sequencing revealed isolation of transformants carrying intact copies the gene clusters was possible. Known Fusarium cytokinin metabolites; fusatin, 8-oxo-fusatin, 8-oxo-isopentenyladenine, fusatinic acid together with cis- and trans-zeatin were detected by liquid chromatography and mass spectrometry, which confirmed gene functionality in F. graminearum. In addition the non-ribosomal lipopeptide products W493 A and B was heterologously produced in similar amounts to that observed in the F. pseudograminearum doner. The Fusarium pan-genome comprises more than 60 uncharacterized putative secondary metabolite gene clusters. We nominate the well-characterized F. graminearum as a heterologous expression platform for Fusarium secondary metabolite gene clusters, and present our experience cloning and introducing gene clusters into this species. We expect the presented methods will inspire future endevours in heterologous production of Fusarium metabolites and potentially aid the production and characterization of novel natural products.

Tissue, urine and blood metabolite signatures of chronic kidney disease in the 5/6 nephrectomy rat model.

INTRODUCTION: Progressive chronic kidney disease (CKD) is an important cause of morbidity and mortality. It has a long asymptomatic phase, where routine blood tests cannot identify early functional losses, and therefore identifying common mechanisms across the many etiologies is an important goal. OBJECTIVES: Our aim was to characterize serum, urine and tissue (kidney, lung, heart, spleen and liver) metabolomics changes in a rat model of CKD. METHODS: A total of 17 male Wistar rats underwent 5/6 nephrectomy, whilst 13 rats underwent sham operation. Urine samples were collected weekly, for 6 weeks; blood was collected at weeks 0, 3 and 6; and tissue samples were collected at week 6. Samples were analyzed on a nuclear magnetic resonance spectroscopy platform with multivariate and univariate data analysis. RESULTS: Changes in several metabolites were statistically significant. Allantoin was affected in all compartments. Renal asparagine, creatine, hippurate and trimethylamine were significantly different; in other tissues creatine, dimethylamine, dimethylglycine, trigonelline and trimethylamine were significant. Benzoate, citrate, dimethylglycine, fumarate, guanidinoacetate, malate, myo-inositol and oxoglutarate were altered in urine or serum. CONCLUSION: Although the metabolic picture is complex, we suggest oxidative stress, the gut-kidney axis, acid-base balance, and energy metabolism as promising areas for future investigation.

Citrate NMR peak irreproducibility in blood samples after reacquisition of spectra.

BACKGROUND: In our metabolomics studies we have noticed that repeated NMR acquisition on the same sample can result in altered metabolite signal intensities. AIMS: To investigate the reproducibility of repeated NMR acquisition on selected metabolites in serum and plasma from two large human metabolomics studies. METHODS: Two peak regions for each metabolite were integrated and changes occurring after reacquisition were correlated. RESULTS: Integral changes were generally small, but serum citrate signals decreased significantly in some samples. CONCLUSIONS: Several metabolite integrals were not reproducible in some of the repeated spectra. Following established protocols, randomising analysis order and biomarker validation are important.

Interactions of a fungal lytic polysaccharide monooxygenase with ß-glucan substrates and cellobiose dehydrogenase.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched ß-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

Characterization of Eight Novel Spiroleptosphols from Fusarium avenaceum.

Chemical analyses of Fusarium avenaceum grown on banana medium resulted in eight novel spiroleptosphols, T1, T2 and U-Z (1-8). The structures were elucidated by a combination of high-resolution mass spectrometric data and 1- and 2-D NMR experiments. The relative stereochemistry was assigned by 1H coupling and NOESY/ROESY experiments. Absolute stereochemistry established for 7 by vibrational circular dichroism was found analogous to that of the putative polyketide spiroleptosphol from Leptosphaeria doliolum.

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor.

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+ These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction.