Narayanan Srividya

Determination of the Line Tension of Giant Vesicles from Pore-Closing Dynamics

Giant vesicles generated from synthetic and natural lipids such as phosphatidylcholines are useful models for understanding mechanical properties of cell membranes. Line tension is the one-dimensional force enabling the closing of transient pores on cell membranes. Transient pores were repeatedly and reproducibly formed on the membrane edge of giant vesicles generated from synthetic and natural phosphatidylcholines employing a nitrogen-pumped coumarin dye laser (440 nm). Line tension was determined at room temperature from closing of these pores that occurred over several seconds when the radius of the vesicle could be considered to be constant. The value of line tension depends on the nature of the lipid for single lipid systems, which, at room temperature, yielded a vesicle bilayer region in the gel, fluid, or mixed gel and fluid phases. The line tension for vesicles generated from phosphatidylcholines with saturated acyl chains of lengths of 12-18 carbon atoms ranges from 1 to 12 pN, exhibiting an increase with chain length. Vesicles generated from the natural Egg-PC, which is a mixture of lipids, are devoid of phase transition and exhibited the largest value of line tension (32 pN). This value is much larger than that estimated from the line tensions of vesicles obtained from lipids with homologous acyl chains. This study, to our knowledge, is the first to employ laser ablation to generate transient pores and determine line tension from the rate of pore closure and demonstrate a relationship between line tension and acyl chain length.

Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase

Significance Terpene synthases catalyze complex, chain length-specific, electrophilic cyclization reactions that constitute the first committed step in the biosynthesis of structurally diverse terpenoids. (4S)-limonene synthase [(4S)-LS] has emerged as a model enzyme for enhancing our comprehension of the reaction cycle of monoterpene (C10) synthases. While the stereochemistry of the cyclization of geranyl diphosphate to (−)-(4S)-limonene has been the subject of several mechanistic studies, the structural basis for the stabilization of carbocation intermediates and the termination of the reaction sequence have remained enigmatic. We present extensive experimental evidence that the aromatic amino acids W324 and H579 play critical roles in the stabilization of intermediate carbocations. A possible function of these residues as the terminal catalytic base is also discussed. Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

Multiple levels of regulation determine monoterpenoid essential oil compositional variation in the mint family.

The genus Mentha has considerable economic importance as a source of essential oils for the flavor, fragrance, and aromatherapy industries (Lange and Ahkami, 2013). The history of commercial mint cultivars is complex and involves various polyploid hybrids. The most widely grown cultivars are essentially sterile, which has impeded the development of genetic resources. The work presented here aims to shed more light into the factors that determine oil composition, which is important for clonal selection strategies.

Integrative Approaches for the Identification and Localization of Specialized Metabolites in Tripterygium Roots

An analysis of the thunder god vine (genus Tripterygium) offers innovative analytical approaches for the identificationand localization of specialized metabolites. Members of the genus Tripterygium are known to contain an astonishing diversity of specialized metabolites. The lack of authentic standards has been an impediment to the rapid identification of such metabolites in extracts. We employed an approach that involves the searching of multiple, complementary chromatographic and spectroscopic data sets against the Spektraris database to speed up the metabolite identification process. Mass spectrometry-based imaging indicated a differential localization of triterpenoids to the periderm and sesquiterpene alkaloids to the cortex layer of Tripterygium roots. We further provide evidence that triterpenoids are accumulated to high levels in cells that contain suberized cell walls, which might indicate a mechanism for storage. To our knowledge, our data provide first insights into the cell type specificity of metabolite accumulation in Tripterygium and set the stage for furthering our understanding of the biological implications of specialized metabolites in this genus.

Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures

Adventitious root cultures provide insights to elucidating the biosynthesis of pharmaceutically relevant diterpenoids in the model genus Tripterygium. Adventitious root cultures were developed from Tripterygium regelii, and growth conditions were optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f, and four to the TPS-b subfamilies. These genes were characterized by heterologous expression in a modular metabolic engineering system in Escherichia coli. Members of the TPS-c subfamily were characterized as copalyl diphosphate (diterpene) synthases, and those belonging to the TPS-e/f subfamily catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis and others with activities as monoterpene synthases. The structural characterization of diterpenoids accumulating in the medium of T. regelii adventitious root cultures, facilitated by searching the Spektraris online spectral database, enabled us to formulate a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with pharmaceutical potential. Considering the significant enrichment of diterpenoids in the culture medium, fast-growing adventitious root cultures may hold promise as a sustainable resource for the large-scale production of triptolide.

Bioenergetics of Monoterpenoid Essential Oil Biosynthesis in Nonphotosynthetic Glandular Trichomes

In nonphotosynthetic glandular trichomes, terpenoid metabolism is powered by oxidative phosphorylation, fermentation and a unique ferredoxin/ferredoxin-NADP+ reductase pair. The commercially important essential oils of peppermint (Mentha × piperita) and its relatives in the mint family (Lamiaceae) are accumulated in specialized anatomical structures called glandular trichomes (GTs). A genome-scale stoichiometric model of secretory phase metabolism in peppermint GTs was constructed based on current biochemical and physiological knowledge. Fluxes through the network were predicted based on metabolomic and transcriptomic data. Using simulated reaction deletions, this model predicted that two processes, the regeneration of ATP and ferredoxin (in its reduced form), exert substantial control over flux toward monoterpenes. Follow-up biochemical assays with isolated GTs indicated that oxidative phosphorylation and ethanolic fermentation were active and that cooperation to provide ATP depended on the concentration of the carbon source. We also report that GTs with high flux toward monoterpenes express, at very high levels, genes coding for a unique pair of ferredoxin and ferredoxin-NADP+ reductase isoforms. This study provides, to our knowledge, the first evidence of how bioenergetic processes determine flux through monoterpene biosynthesis in GTs.

Generation and Functional Evaluation of Designer Monoterpene Synthases

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes.

Rapid purification of gram quantities of β-sitosterol from a commercial phytosterol mixture

Backgroundβ-Sitosterol, a plant sterol or phytosterol, has commercial uses in the nutraceutical and pharmaceutical industries, but is also employed frequently in biological research. Phytosterols always accumulate as mixtures, and obtaining highly pure β-sitosterol in larger quantities for biological assays has been a challenge.FindingsAn improved method for the rapid purification of β-sitosterol from a commercial phytosterol extract is presented. Fractional crystallization of soybean oil yielded a soluble and an insoluble fraction. β-Sitosterol was purified by silica gel and Na-Y zeolite chromatography.ConclusionThe rapid and cost effective three-step purification described here afforded β-sitosterol in gram quantities with high purity (>92%) and yield (>22%).