150 research outputs found
Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae
The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs. Our previous discovery of GAO activity conserved across all major subfamilies of Asteraceae, including the phylogenetically basal lineage of Barnadesioideae, prompted further investigation of the presence of the gateway GAS in Barnadesioideae. Herein we isolated two terpene synthases (BsGAS1/BsGAS2) from the basal Barnadesia spinosa (Barnadesioideae) that displayed robust GAS activity when reconstituted in yeast and characterized in vitro. Despite the apparent lack of STLs in the Barnadesioideae, this work unambiguously confirms the presence of GAS in the basal genera of the Asteraceae. Phylogenetic analysis reveals that the two BsGASs fall into two distinct clades of the Asteraceae's GASs, and BsGAS1 clade is only retained in the evolutionary closer Cichorioideae subfamily, implicating BsGAS2 is likely the ancestral base of most GASs found in the lineages outside the Barnadesioideae. Taken together, these results show the enzymatic capacities of GAS and GAO emerged prior to the subsequent radiation of STL-producing Asteraceae subfamilies
Mobilization of pro-inflammatory lipids in obese Plscr3-deficient mice
Metabolic profiling of mice deficient in phospholipid scramblase 3 reveals a possible molecular link between obesity and inflammation
Predictability of evolutionary trajectories in fitness landscapes
Experimental studies on enzyme evolution show that only a small fraction of
all possible mutation trajectories are accessible to evolution. However, these
experiments deal with individual enzymes and explore a tiny part of the fitness
landscape. We report an exhaustive analysis of fitness landscapes constructed
with an off-lattice model of protein folding where fitness is equated with
robustness to misfolding. This model mimics the essential features of the
interactions between amino acids, is consistent with the key paradigms of
protein folding and reproduces the universal distribution of evolutionary rates
among orthologous proteins. We introduce mean path divergence as a quantitative
measure of the degree to which the starting and ending points determine the
path of evolution in fitness landscapes. Global measures of landscape roughness
are good predictors of path divergence in all studied landscapes: the mean path
divergence is greater in smooth landscapes than in rough ones. The
model-derived and experimental landscapes are significantly smoother than
random landscapes and resemble additive landscapes perturbed with moderate
amounts of noise; thus, these landscapes are substantially robust to mutation.
The model landscapes show a deficit of suboptimal peaks even compared with
noisy additive landscapes with similar overall roughness. We suggest that
smoothness and the substantial deficit of peaks in the fitness landscapes of
protein evolution are fundamental consequences of the physics of protein
folding.Comment: 14 pages, 7 figure
The evolutionary paths towards complexity: a metabolic perspective
As sessile organisms, land plants have exploited their metabolic systems to produce a panoply of structurally and functionally diverse natural chemicals and polymers to adapt to challenging ecosystems. Many of these core and specialized metabolites confer chemical shields against a multitude of abiotic stresses, while others play important roles in plants' interactions with their biotic environments. Plant specialized metabolites can be viewed as complex traits in the sense that the biosynthesis of these molecules typically requires multistep metabolic pathways comprising numerous specific enzymes belonging to diverse protein fold families. Resolving the evolutionary trajectories underlying the emergence of these specialized metabolic pathways will impact a fundamental question in biology – how do complex traits evolve in a Darwinian fashion? Here, I discuss several general patterns observed in rapidly evolving specialized metabolic systems in plants, and surmise mechanistic features at enzyme, pathway and organismal levels that rationalize the remarkable malleability of these systems through stepwise evolution. Future studies, focused on fine sampling of metabolic enzymes and pathways in phylogenetically related plant species, or employing directed evolution strategies in synthetic systems, will significantly broaden our perspective on how biological complexity arises at the metabolic level.Howard Hughes Medical InstitutePioneer Foundation (Postdoctoral Fellowship
Identification and biosynthesis of acylphloroglucinols in Hypericum gentianoides
Species of the genus Hypericum contain a rich array of unusual polyketides, however, only a small proportion of the over 450 Hypericum species, other than the popular medicinal supplement St. John’s Wort (H. perforatum), have even been chemically characterized. H. gentianoides, a small annual used medicinally by Cherokee Americans, contains bioactive acylphloroglucinols. Here, we identify acylphloroglucinol constituents of H. gentianoides and determine a potential pathway to their synthesis. Liquid chromatography/electrospray ionization-mass spectrometry (LC/ESIMS) and HPLC-UV indicate that the level of accumulation and profile of acylphloroglucinols in H. gentianoides vary little seasonally when grown in a greenhouse, but do vary with development and are highly dependent on the accession, highlighting the importance of the selection of plant material for study. We identify the chemical structures of the nine prevalent polyketides, based on LC/ESI-MS and hybrid quadrupole orthogonal time-of-flight mass (Q-TOF) spectrometry; these metabolites include one monomeric phlorisobutyrophenone (PIB) derivative and eight dimeric acylphloroglucinols. Q-TOF spectrometry was used to identify eight additional PIB derivatives that were not detected by LC/ESI-MS. These data lead us to propose that diacylphloroglucinols are synthesized via modification of PIB to yield diverse phloroglucinol and filicinic acids moieties, followed by dimerization of a phloroglucinol and a filicinic acid monomer to yield the observed complement of diacylphloroglucinols. The metabolomics data from H. gentianoides are accessible in PMR (http://www.metnetdb.org/pmr), a public metabolomics database with analysis software for plants and microbial organisms
Exploring the Complexity of the HIV-1 Fitness Landscape
Although fitness landscapes are central to evolutionary theory, so far no biologically realistic examples for large-scale fitness landscapes have been described. Most currently available biological examples are restricted to very few loci or alleles and therefore do not capture the high dimensionality characteristic of real fitness landscapes. Here we analyze large-scale fitness landscapes that are based on predictive models for in vitro replicative fitness of HIV-1. We find that these landscapes are characterized by large correlation lengths, considerable neutrality, and high ruggedness and that these properties depend only weakly on whether fitness is measured in the absence or presence of different antiretrovirals. Accordingly, adaptive processes on these landscapes depend sensitively on the initial conditions. While the relative extent to which mutations affect fitness on their own (main effects) or in combination with other mutations (epistasis) is a strong determinant of these properties, the fitness landscape of HIV-1 is considerably less rugged, less neutral, and more correlated than expected from the distribution of main effects and epistatic interactions alone. Overall this study confirms theoretical conjectures about the complexity of biological fitness landscapes and the importance of the high dimensionality of the genetic space in which adaptation takes place
Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases
Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases
Chapter Two – Automating Gene Library Synthesis by Structure-Based Combinatorial Protein Engineering: Examples from Plant Sesquiterpene Synthases
Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of functionally important mutations. Through these examples, the principles of the technique are illustrated and the suitability of automating various aspects of the procedure for given applications are discussed
Hepatic Storage and Biliary Transport Maximum of Taurocholate and Taurochenodeoxycholate in the Dog
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