Multiproduct Terpene Synthases:Catalytic Promiscuity and Cyclization of Substrate Geometric Isomers

Terpenes constitute the largest and the most diverse class of natural products. The wealth of terpenes can be attributed to highly promiscuous enzymes called terpene synthases. Apart from single product enzymes, there also exist multiproduct terpene synthases generating a bouquet of acyclic and cyclic products. This thesis is aimed at using substrate analogs to study catalytic promiscuity and mechanistic pathways of multiproduct terpene synthases. The mechanistic details of reaction cascade in TPS4 and TPS5 from Zea mays were evaluated by using isotope sensitive branching. Labeled substrates with deuterium atoms completely surrounding the key cationic intermediates were used as metabolic probes. Apart from the confirmation of mechanistic cascade, kinetic isotope effects on terminating deprotonations led to an enhanced formation of alcohols over olefinic products. Deuterium labeled (2Z)-substrate geometric isomers, mimicking the nerolidyl diphosphate intermediate with TPS4 and TPS5 generated the same product profile but with increased cyclic products. Major increase in enzymatic turnover was also observed with (2Z)-substrates emphasize the rate limiting effect of the initial isomerization step. In contrast, MtTPS5 from Medicago truncatula showed a new product profile with majority of products formed via a C1-C11 ring closure to the humulyl cation over the natural cadalane skeleton. This demonstrates the possibility of using substrate geometry as tool to generate novel products. The structural characteristics of multiproduct terpene synthases remain unresolved due to absence of defined crystal structures. With some initial signs of success as co-crystallization candidates, 3-bromo analogs of substrates were found to be potent competitive inhibitors of MtTPS5 and other terpene synthases. Consequently with this work, catalytic promiscuity of multiproduct terpene synthases can be employed to design better biocatalysts with improved turnover and generate novel products

Alternate cyclization cascade initiated by substrate isomer in multiproduct terpene synthase from Medicago truncatula

Promiscuity of terpene synthases results in the enormous diversity of terpenes found in nature. Multiproduct sesquiterpene synthase MtTPS5 isolated from Medicago truncatula generates 27 optically pure products from its natural substrate (2E,6E)-farnesyl diphosphate (FDP). In order to study the promiscuity of MtTPS5, (2Z,6E)-FDP an analogue of presumptive reaction intermediates from natural reaction cascade was utilized as a substrate. This stereoisomer induced a novel cyclization pathway leading to sesquiterpenes based on humulane, amorphene and himachalane skeletons. Interestingly, none of these products matched with those observed on incubation of MtTPS5 with natural (2E,6E)- FDP. Further determination of absolute configuration of each product helped rebuild the stereochemical route of the reaction cascade. Interestingly, the presence of only one enantiomer of each product was observed indicating the highly stereospecific nature of the enzymatic reaction. Substrate promiscuity of terpene synthases provides organism access to novel chemical bouquets of high optical purity by utilizing existing enzymes. Presence of this mechanism was indicated by the presence of these alternate products in natural herbivore-induced volatiles of Medicago truncatula

Enhanced structural diversity in terpenoid biosynthesis: enzymes, substrates and cofactors

The enormous diversity of terpenes found in nature is generated by enzymes known as terpene synthases, or cyclases. Some are also known for their ability to convert a single substrate into multiple products. This review comprises monoterpene and sesquiterpene synthases that are multiproduct in nature along with the regulation factors that can alter the product specificity of multiproduct terpene synthases without genetic mutations. Variations in specific assay conditions with focus on shifts in product specificity based on change in metal cofactors, assay pH and substrate geometry are described. Alterations in these simple cellular conditions provide the organism with enahnced chemodiversity without investing into new enzymatic architecture. This versatility to modulate product diversity grants organisms, especially immobile ones like plants with access to an enhanced defensive repertoire by simply altering cofactors, pH level and substrate geometry

Fluorinated Analogs of Organosulfur Compounds from Garlic (Allium sativum): Synthesis, Chemistry and Anti-Angiogenesis and Antithrombotic Studies

We describe the synthesis, reactivity, and antithrombotic and anti-angiogenesis activity of difluoroallicin (S-(2-fluoroallyl) 2-fluoroprop-2-ene-1-sulfinothioate) and S-2-fluoro-2-propenyl-l-cysteine, both easily prepared from commercially available 3-chloro-2-fluoroprop-1-ene, as well as the synthesis of 1,2-bis(2-fluoroallyl)disulfane, 5-fluoro-3-(1-fluorovinyl)-3,4-dihydro-1,2-dithiin, trifluoroajoene ((E,Z)-1-(2-fluoro-3-((2-fluoroallyl)sulfinyl)prop-1-en-1-yl)-2-(2-fluoroallyl)disulfane), and a bis(2-fluoroallyl)polysulfane mixture. All tested organosulfur compounds demonstrated effective inhibition of either FGF or VEG-mediated angiogenesis (anti-angiogenesis activity) in the chick chorioallantoic membrane (CAM) or the mouse Matrigel® models. No embryo mortality was observed. Difluoroallicin demonstrated greater inhibition (p < 0.01) versus organosulfur compounds tested. Difluoroallicin demonstrated dose-dependent inhibition of angiogenesis in the mouse Matrigel® model, with maximal inhibition at 0.01 mg/implant. Allicin and difluoroallicin showed an effective antiplatelet effect in suppressing platelet aggregation compared to other organosulfur compounds tested. In platelet/fibrin clotting (anti-coagulant activity), difluoroallicin showed concentration-dependent inhibition of clot strength compared to allicin and the other organosulfur compounds tested

Correction: Block et al. Fluorinated Analogs of Organosulfur Compounds from Garlic (<i>Allium sativum</i>): Synthesis, Chemistry and Anti-Angiogenesis and Antithrombotic Studies. <i>Molecules</i> 2017, <i>22</i>, 2081

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Trifluoroselenomethionine - a new unnatural amino acid

Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N-(tert-butoxycarbonyl)-l-aspartic acid tert-butyl ester. TFSeM shows enhanced methioninase-induced cytotoxicity, relative to selenomethionine (SeM), toward HCT-116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase