Complete stereoinversion of L-tryptophan by a fungal single module nonribosomal peptide synthetase

Single-module nonribosomal peptide synthetases (NRPSs) and NRPS-like enzymes activate and transform carboxylic acids in both primary and secondary metabolism and are of great interest due to their biocatalytic potentials. The single-module NRPS IvoA is essential for fungal pigment biosynthesis. Here, we show that IvoA catalyzes ATP-dependent unidirectional stereoinversion of l-tryptophan to d-tryptophan with complete conversion. While the stereoinversion is catalyzed by the epimerization (E) domain, the terminal condensation (C) domain stereoselectively hydrolyzes d-tryptophanyl-S-phosphopantetheine thioester and thus represents a noncanonical C domain function. Using IvoA, we demonstrate a biocatalytic stereoinversion/deracemization route to access a variety of substituted d-tryptophan analogs in high enantiomeric excess

Geodynamic implications for zonal and meridional isotopic patterns across the northern Lau and North Fiji Basins

We present new Sr-Nd-Pb-Hf-He isotopic data for sixty-five volcanic samples from the northern Lau and North Fiji Basin. This includes forty-seven lavas obtained from forty dredge sites spanning an east-west transect across the Lau and North Fiji basins, ten ocean island basalt (OIB)-type lavas collected from seven Fijian islands, and eight OIB lavas sampled on Rotuma. For the first time we are able to map clear north-south and east-west geochemical gradients in 87Sr/86Sr across the northern Lau and North Fiji Basins: lavas with the most geochemically enriched radiogenic isotopic signatures are located in the northeast Lau Basin, while signatures of geochemical enrichment are diminished to the south and west away from the Samoan hotspot. Based on these geochemical patterns and plate reconstructions of the region, these observations are best explained by the addition of Samoa, Rurutu, and Rarotonga hotspot material over the past 4 Ma. We suggest that underplated Samoan material has been advected into the Lau Basin over the past ∼4 Ma. As the slab migrated west (and toward the Samoan plume) via rollback over time, younger and hotter (and therefore less viscous) underplated Samoan plume material was entrained. Thus, entrainment efficiency of underplated plume material was enhanced, and Samoan plume signatures in the Lau Basin became stronger as the trench approached the Samoan hotspot. The addition of subducted volcanoes to the Cook-Austral Volcanic Lineament material, first from the Rarotonga hotspot, then followed by the Rurutu hotspot, contributes to the extreme geochemical signatures observed in the northeast Lau Basin

Eumalacostracan phylogeny and total evidence: limitations of the usual suspects

<p>Abstract</p> <p>Background</p> <p>The phylogeny of Eumalacostraca (Crustacea) remains elusive, despite over a century of interest. Recent morphological and molecular phylogenies appear highly incongruent, but this has not been assessed quantitatively. Moreover, 18S rRNA trees show striking branch length differences between species, accompanied by a conspicuous clustering of taxa with similar branch lengths. Surprisingly, previous research found no rate heterogeneity. Hitherto, no phylogenetic analysis of all major eumalacostracan taxa (orders) has either combined evidence from multiple loci, or combined molecular and morphological evidence.</p> <p>Results</p> <p>We combined evidence from four nuclear ribosomal and mitochondrial loci (18S rRNA, 28S rRNA, 16S rRNA, and cytochrome <it>c </it>oxidase subunit I) with a newly synthesized morphological dataset. We tested the homogeneity of data partitions, both in terms of character congruence and the topological congruence of inferred trees. We also performed Bayesian and parsimony analyses on separate and combined partitions, and tested the contribution of each partition. We tested for potential long-branch attraction (LBA) using taxon deletion experiments, and with relative rate tests. Additionally we searched for molecular polytomies (spurious clades). Lastly, we investigated the phylogenetic stability of taxa, and assessed their impact on inferred relationships over the whole tree. We detected significant conflict between data partitions, especially between morphology and molecules. We found significant rate heterogeneity between species for both the 18S rRNA and combined datasets, introducing the possibility of LBA. As a test case, we showed that LBA probably affected the position of Spelaeogriphacea in the combined molecular evidence analysis. We also demonstrated that several clades, including the previously reported and surprising clade of Amphipoda plus Spelaeogriphacea, are 'supported' by zero length branches. Furthermore we showed that different sets of taxa have the greatest impact upon the relationships within molecular versus morphological trees.</p> <p>Conclusion</p> <p>Rate heterogeneity and conflict between data partitions mean that existing molecular and morphological evidence is unable to resolve a well-supported eumalacostracan phylogeny. We believe that it will be necessary to look beyond the most commonly utilized sources of data (nuclear ribosomal and mitochondrial sequences) to obtain a robust tree in the future.</p

Efficient inference and identifiability analysis for differential equation models with random parameters

Heterogeneity is a dominant factor in the behaviour of many biological processes. Despite this, it is common for mathematical and statistical analyses to ignore biological heterogeneity as a source of variability in experimental data. Therefore, methods for exploring the identifiability of models that explicitly incorporate heterogeneity through variability in model parameters are relatively underdeveloped. We develop a new likelihood-based framework, based on moment matching, for inference and identifiability analysis of differential equation models that capture biological heterogeneity through parameters that vary according to probability distributions. As our novel method is based on an approximate likelihood function, it is highly flexible; we demonstrate identifiability analysis using both a frequentist approach based on profile likelihood, and a Bayesian approach based on Markov-chain Monte Carlo. Through three case studies, we demonstrate our method by providing a didactic guide to inference and identifiability analysis of hyperparameters that relate to the statistical moments of model parameters from independent observed data. Our approach has a computational cost comparable to analysis of models that neglect heterogeneity, a significant improvement over many existing alternatives. We demonstrate how analysis of random parameter models can aid better understanding of the sources of heterogeneity from biological data.Comment: Minor changes to text. Additional results in supplementary material. Additional statistics regarding results given in main and supplementary materia

Acyl-chain elongation drives ketosynthase substrate selectivity in trans-acyltransferase polyketide synthases

Type I modular polyketide synthases (PKSs), responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl-acyl carrier protein, yielding an extended β-ketoacyl chain. To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. We submitted six KS domains from the trans-acyl transferase PKSs to a mass spectrometry-basedelongation assay, and identified higher substrat selectivity in the elongating step than in preceding acylation. A close correspondence between observed KS selectivity and that predicted by phylogenetic analysis was seen. Our findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates

Structural characterization of cephaeline binding to the eukaryotic ribosome using Cryo-Electron Microscopy

The eukaryotic ribosome is emerging as a promising target against human pathogens, includ- ing amoeba, protozoans, and fungi. Among the eukaryotic-specific families of inhibitors, al- kaloids are known to bind to the eukaryotic ribosome and inhibit translocation. However, these inhibitors have varying medical indications and toxicity to humans. Structural information is available for only two of them, cryptopleurine and emetine. Aim. In our work, we aimed to elucidate the binding mechanism of another alkaloid, cephaeline, to the eukaryotic ribosome. Methods. We used cryogenic electron microscopy and cell-free assays to reveal its mechanism of action. Results. Our results indicate that cephaeline binds to the E-tRNA binding site on the small subunit of the eukaryotic ribosome. Similar to emetine, cephaeline forms a stacking interaction with G889 of 18S rRNA and L132 of the protein uS11. We propose the hypothesis of cephaeline specificity to eukaryotes by comparing the interaction pattern of cephaeline with other inhibitors binding to the E-site of the mRNA tunnel. Conclusions. The high-resolution structure of ribosome-bound cephaeline (2.45 Å) allowed us to precisely determine the in- hibitor’s position in the binding site, which holds potential for the development of the next generation of drugs targeting the mRNA tunnel of the ribosome

Structural characterization of cephaeline binding to the eukaryotic ribosome using Cryo-Electron Microscopy

The eukaryotic ribosome is emerging as a promising target against human pathogens, includ- ing amoeba, protozoans, and fungi. Among the eukaryotic-specific families of inhibitors, al- kaloids are known to bind to the eukaryotic ribosome and inhibit translocation. However, these inhibitors have varying medical indications and toxicity to humans. Structural information is available for only two of them, cryptopleurine and emetine. Aim. In our work, we aimed to elucidate the binding mechanism of another alkaloid, cephaeline, to the eukaryotic ribosome. Methods. We used cryogenic electron microscopy and cell-free assays to reveal its mechanism of action. Results. Our results indicate that cephaeline binds to the E-tRNA binding site on the small subunit of the eukaryotic ribosome. Similar to emetine, cephaeline forms a stacking interaction with G889 of 18S rRNA and L132 of the protein uS11. We propose the hypothesis of cephaeline specificity to eukaryotes by comparing the interaction pattern of cephaeline with other inhibitors binding to the E-site of the mRNA tunnel. Conclusions. The high-resolution structure of ribosome-bound cephaeline (2.45 Å) allowed us to precisely determine the in- hibitor’s position in the binding site, which holds potential for the development of the next generation of drugs targeting the mRNA tunnel of the ribosome

Structural characterization of cephaeline binding to the eukaryotic ribosome using Cryo-Electron Microscopy

The eukaryotic ribosome is emerging as a promising target against human pathogens, includ- ing amoeba, protozoans, and fungi. Among the eukaryotic-specific families of inhibitors, al- kaloids are known to bind to the eukaryotic ribosome and inhibit translocation. However, these inhibitors have varying medical indications and toxicity to humans. Structural information is available for only two of them, cryptopleurine and emetine. Aim. In our work, we aimed to elucidate the binding mechanism of another alkaloid, cephaeline, to the eukaryotic ribosome. Methods. We used cryogenic electron microscopy and cell-free assays to reveal its mechanism of action. Results. Our results indicate that cephaeline binds to the E-tRNA binding site on the small subunit of the eukaryotic ribosome. Similar to emetine, cephaeline forms a stacking interaction with G889 of 18S rRNA and L132 of the protein uS11. We propose the hypothesis of cephaeline specificity to eukaryotes by comparing the interaction pattern of cephaeline with other inhibitors binding to the E-site of the mRNA tunnel. Conclusions. The high-resolution structure of ribosome-bound cephaeline (2.45 Å) allowed us to precisely determine the in- hibitor’s position in the binding site, which holds potential for the development of the next generation of drugs targeting the mRNA tunnel of the ribosome

Structural characterization of cephaeline binding to the eukaryotic ribosome using Cryo-Electron Microscopy

The eukaryotic ribosome is emerging as a promising target against human pathogens, includ- ing amoeba, protozoans, and fungi. Among the eukaryotic-specific families of inhibitors, al- kaloids are known to bind to the eukaryotic ribosome and inhibit translocation. However, these inhibitors have varying medical indications and toxicity to humans. Structural information is available for only two of them, cryptopleurine and emetine. Aim. In our work, we aimed to elucidate the binding mechanism of another alkaloid, cephaeline, to the eukaryotic ribosome. Methods. We used cryogenic electron microscopy and cell-free assays to reveal its mechanism of action. Results. Our results indicate that cephaeline binds to the E-tRNA binding site on the small subunit of the eukaryotic ribosome. Similar to emetine, cephaeline forms a stacking interaction with G889 of 18S rRNA and L132 of the protein uS11. We propose the hypothesis of cephaeline specificity to eukaryotes by comparing the interaction pattern of cephaeline with other inhibitors binding to the E-site of the mRNA tunnel. Conclusions. The high-resolution structure of ribosome-bound cephaeline (2.45 Å) allowed us to precisely determine the in- hibitor’s position in the binding site, which holds potential for the development of the next generation of drugs targeting the mRNA tunnel of the ribosome