The Experts below are selected from a list of 168 Experts worldwide ranked by ideXlab platform
Vladimir I Kalinin - One of the best experts on this subject based on the ideXlab platform.
-
kurilosides a1 a2 c1 d e and f triterpene glycosides from the far eastern sea cucumber thyonidium duasmodactyla kurilensis levin structures with unusual non holostane aglycones and cytotoxicities
Marine Drugs, 2020Co-Authors: Alexandra S Silchenko, Sergey A Avilov, Pavel S. Dmitrenok, Anatoly I Kalinovsky, Pelageya V Andrijaschenko, Roman S Popov, Ekaterina A Chingizova, Vladimir I KalininAbstract:Six new monosulfated triterpene tetra-, penta- and hexaosides, namely, the kurilosides A1 (1), A2 (2), C1 (3), D (4), E (5) and F (6), as well as the known earlier kuriloside A (7), having unusual non-holostane aglycones without lactone, have been isolated from the sea cucumber Thyonidium (= Duasmodactyla) kurilensis (Levin) (Cucumariidae, Dendrochirotida), collected in the Sea of Okhotsk near Onekotan Island from a depth of 100 m. Structures of the glycosides were established by 2D NMR spectroscopy and HR-ESI mass spectrometry. Kurilosides of the groups A and E contain carbohydrate moieties with a rare architecture (a pentasaccharide branched by C(4) Xyl1), differing from each other in the second monosaccharide residue (Quinovose or glucose, correspondingly); kurilosides of the group C are characterized by a unique tetrasaccharide branched by a C(4) Xyl1 sugar chain; and kurilosides of the groups D and F are hexaosides differing from each other in the presence of an O-methyl group in the fourth (terminal) sugar unit. All these glycosides contain a sulfate group at C-6 of the glucose residue attached to C-4 Xyl1 and the non-holostane aglycones have a 9(11) double bond and lack γ-lactone. The cytotoxic activities of compounds 1–7 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells and erythrocytes were studied. Kuriloside A1 (1) was the most active compound in the series, demonstrating strong cytotoxicity against the erythrocytes and JB-6 cells and a moderate effect against Neuro 2a cells.
-
structures and biological activities of cladolosides c3 e1 e2 f1 f2 g h1 and h2 eight triterpene glycosides from the sea cucumber cladolabes schmeltzii with one known and four new carbohydrate chains
Carbohydrate Research, 2015Co-Authors: Alexandra S Silchenko, Sergey A Avilov, Anatoly I Kalinovsky, Pelageya V Andryjaschenko, Ekaterina A Yurchenko, Igor Yu Dolmatov, Vladimir I KalininAbstract:Eight new nonsulfated triterpene glycosides, cladolosides C3(1), E1(2), E2(3), F1(4), F2(5), G(6), H1(7) and H2(8) have been isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida) collected in the Vietnamese shallow waters. The structures of the glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. Glycosides 2, 3, 4, and 5 have pentasaccharide branched carbohydrate moieties and differ from each other by monosaccharide compositions and aglycone structures. At that, glycosides 2 and 3 contain three xylose, one 3-O-methyl-glucose and one Quinovose residues, while glycosides 4 and 5 have two Quinovose, two xylose and one 3-O-methyl-glucose residues. Compounds 1 and 6-8 are hexaosides differing from each other by aglycone structures and by the fifth monosaccharide residue, which proved to be glucose in cladoloside C3(1), xylose in cladoloside G(6) and Quinovose in cladolosides H1(7) and H2(8). The presence of Quinovose residue in the fifth position, as in 4, 5, 7 and 8 has never been earlier found in carbohydrate chains of triterpene glycosides from sea cucumbers. The carbohydrate chains with xylose in the fifth position of pentaosides and hexaosides are also very unusual for holothurious glycosides. All the substances demonstrate strong or moderate cytotoxic and hemolytic effects with hexaosides being more active than the corresponding pentaosides. Peculiarities of the biosynthesis and biochemical evolution of glycosides of this type are discussed.
-
triterpene glycosides of sea cucumbers holothuroidea echinodermata as taxonomic markers
Natural Product Communications, 2015Co-Authors: Vladimir I Kalinin, Alexandra S Silchenko, Sergey A Avilov, Valentin A StonikAbstract:Triterpene glycosides are characteristic metabolites of sea cucumbers (Holothurioidea, Echinodermata). The majority of the glycosides belong to the holostane type {lanostane derivatives with an 18(20)-lactone}. Carbohydrate chains of these glycosides contain xylose, glucose, Quinovose, 3-O-methylglucose, and, rarely, 3-O-methylxylose, 3-O-methylglucuronic acid, 3-O-methylQuinovose, and 6-O-acetyl-glucose. The glycosides are specific for genera, groups of genera and even for species. The advantages and problems in the use of triterpene glycosides as taxonomic markers in the systematics of sea cucumbers are discussed.
-
immunomodulatory and anticancer activity of sea cucumber triterpene glycosides
Studies in natural products chemistry, 2014Co-Authors: Dmitry L Aminin, Evgeny A Pislyagin, Ekaterina S Menchinskaya, Alexandra S Silchenko, Sergey A Avilov, Vladimir I KalininAbstract:Abstract The triterpene glycosides are composed of a carbohydrate chain and triterpene aglycone and are widely distributed in sea cucumbers ( Holothurioidea , Echinodermata ). Most aglycones have 18(20)-lactones and belong to the holostane type. Carbohydrate chains of sea cucumber glycosides have from two to six monosaccharide residues including xylose, Quinovose, glucose, and 3- O -methylglucose and sometimes 3- O -methylxylose, 3- O -methylQuinovose, 3- O -methylglucuronic acid, and 6- O -acetylglucose. They may contain one, two, or three sulfate groups. At the milli- and micromolar concentrations, sea cucumber glycosides show hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. The basis of membranotropic action of the glycosides is their ability to attach to cell biomembranes and form nonselective ion-conducting complexes with 5(6)-nonsaturated sterol components of cell membranes, preferably with cholesterol. Such sterol/saponin interactions result in an efflux of some ions, nucleotides, and peptides, disrupting ion homeostasis and osmolarity followed by lysis and cell death. Some sea cucumber glycosides show an immunostimulatory effect at subtoxic nanomolar concentrations. Incubation of immune cells with the glycosides induces their activation resulting in an increase of immune cell adhesion on an extracellular matrix, enhancement of cell spreading and motility, increase of macrophage lysosomal activity, ROS formation, and phagocytic activity. The most effective immunostimulants are monosulfated glycosides, whereas di- and trisulfated glycosides are immunodepressants. Injection of subtoxic doses of some glycosides induces an increase in the number of antibody-producing plaque-forming cells in mouse spleens, an increase in the number, size and acidity of lysosomes of peritoneal macrophages, and increase of phagocytic index, resulting in heightened resistance by host animals against bacterial infections. Proteomic methods have demonstrated that the mechanism of immunomodulatory action of some sea cucumber glycosides on mouse splenocytes includes regulation of the expression of some proteins involved in the formation of cellular immune response. These glycosides regulate the expression of proteins associated with lysosome maturation, activation and merging, phagocytosis, cytoskeletal reorganization, cell adhesion, mobility, and proliferation of immune cells. It was shown that glycosides moderately induce production of some cytokines, restore the level of some CD markers of lymphocytes, increase bactericidal activity of leukocytes, and induce a significant increase in mouse resistance to lethal doses of some pathogenic microorganisms and radiation. Cytotoxic activity of sea cucumber glycosides against different types of cells and cell lines, including human tumor cell lines, has been studied for many years. These studies have shown the triterpene glycosides block egg cleavage and development of sea urchin embryos, suppress the proliferation of various human tumor cell lines in vitro , possess antiangiogenic effect, and cause cancer cell cycle arrest. Several sea cucumber glycosides are reported to induce tumor cell apoptosis in vitro and more importantly, IP administration in rodents of solutions of some sea cucumber triterpene glycosides show significant reduction of both tumor burden and metastasis. Recently, it was found that the new immunomodulatory lead compound, Cumaside, based on the holothurian triterpene glycoside, cucumarioside A 2 -2, demonstrates inhibition of tumor initiation and proliferation, in vivo and exhibits significant synergy with 5-fluorouracil.
-
triterpene glycosides from antarctic sea cucumbers iv turquetoside a a 3 o methylQuinovose containing disulfated tetraoside from the sea cucumber staurocucumis turqueti vaney 1906 cucumaria spatha
Biochemical Systematics and Ecology, 2013Co-Authors: Alexandra S Silchenko, Sergey A Avilov, Vladimir I Kalinin, Anatoly I Kalinovsky, Pelageya V Andryjashchenko, Sergi Taboada, Conxita AvilaAbstract:Abstract A novel triterpene holostane disulfated tetrasaccharide olygoglycoside, turquetoside A, having a rare terminal 3- O -methyl-D-Quinovose, was isolated from the Antarctic sea cucumber Staurocucumis turqueti . Its structure was elucidated using 2D NMR procedures (HMBC, HSQC, ROESY) and mass-spectrometry. The occurrence of 3- O -methyl-D-Quinovose in triterpene glycosides in S. turqueti and in the congeneric Antarctic sea cucumber Staurocucumis liouvillei suggests that the presence of this sugar in carbohydrate chains of triterpene glycosides is a taxonomical character of the genus Staurocucumis .
Alexandra S Silchenko - One of the best experts on this subject based on the ideXlab platform.
-
kurilosides a1 a2 c1 d e and f triterpene glycosides from the far eastern sea cucumber thyonidium duasmodactyla kurilensis levin structures with unusual non holostane aglycones and cytotoxicities
Marine Drugs, 2020Co-Authors: Alexandra S Silchenko, Sergey A Avilov, Pavel S. Dmitrenok, Anatoly I Kalinovsky, Pelageya V Andrijaschenko, Roman S Popov, Ekaterina A Chingizova, Vladimir I KalininAbstract:Six new monosulfated triterpene tetra-, penta- and hexaosides, namely, the kurilosides A1 (1), A2 (2), C1 (3), D (4), E (5) and F (6), as well as the known earlier kuriloside A (7), having unusual non-holostane aglycones without lactone, have been isolated from the sea cucumber Thyonidium (= Duasmodactyla) kurilensis (Levin) (Cucumariidae, Dendrochirotida), collected in the Sea of Okhotsk near Onekotan Island from a depth of 100 m. Structures of the glycosides were established by 2D NMR spectroscopy and HR-ESI mass spectrometry. Kurilosides of the groups A and E contain carbohydrate moieties with a rare architecture (a pentasaccharide branched by C(4) Xyl1), differing from each other in the second monosaccharide residue (Quinovose or glucose, correspondingly); kurilosides of the group C are characterized by a unique tetrasaccharide branched by a C(4) Xyl1 sugar chain; and kurilosides of the groups D and F are hexaosides differing from each other in the presence of an O-methyl group in the fourth (terminal) sugar unit. All these glycosides contain a sulfate group at C-6 of the glucose residue attached to C-4 Xyl1 and the non-holostane aglycones have a 9(11) double bond and lack γ-lactone. The cytotoxic activities of compounds 1–7 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells and erythrocytes were studied. Kuriloside A1 (1) was the most active compound in the series, demonstrating strong cytotoxicity against the erythrocytes and JB-6 cells and a moderate effect against Neuro 2a cells.
-
in vitro and in vivo anti leukemic effects of cladoloside c 2 are mediated by activation of fas ceramide synthase 6 p38 kinase c jun nh 2 terminal kinase caspase 8
Oncotarget, 2018Co-Authors: Seonghoon Yun, Alexandra S Silchenko, Valentin A Stonik, Eunhye Sim, Sangheum Han, Taerang Kim, Jinyeong Han, Jinsook Jeong, Sunghyun Kim, Jooin ParkAbstract:We previously demonstrated that the Quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model. Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.
-
structures and biological activities of cladolosides c3 e1 e2 f1 f2 g h1 and h2 eight triterpene glycosides from the sea cucumber cladolabes schmeltzii with one known and four new carbohydrate chains
Carbohydrate Research, 2015Co-Authors: Alexandra S Silchenko, Sergey A Avilov, Anatoly I Kalinovsky, Pelageya V Andryjaschenko, Ekaterina A Yurchenko, Igor Yu Dolmatov, Vladimir I KalininAbstract:Eight new nonsulfated triterpene glycosides, cladolosides C3(1), E1(2), E2(3), F1(4), F2(5), G(6), H1(7) and H2(8) have been isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida) collected in the Vietnamese shallow waters. The structures of the glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. Glycosides 2, 3, 4, and 5 have pentasaccharide branched carbohydrate moieties and differ from each other by monosaccharide compositions and aglycone structures. At that, glycosides 2 and 3 contain three xylose, one 3-O-methyl-glucose and one Quinovose residues, while glycosides 4 and 5 have two Quinovose, two xylose and one 3-O-methyl-glucose residues. Compounds 1 and 6-8 are hexaosides differing from each other by aglycone structures and by the fifth monosaccharide residue, which proved to be glucose in cladoloside C3(1), xylose in cladoloside G(6) and Quinovose in cladolosides H1(7) and H2(8). The presence of Quinovose residue in the fifth position, as in 4, 5, 7 and 8 has never been earlier found in carbohydrate chains of triterpene glycosides from sea cucumbers. The carbohydrate chains with xylose in the fifth position of pentaosides and hexaosides are also very unusual for holothurious glycosides. All the substances demonstrate strong or moderate cytotoxic and hemolytic effects with hexaosides being more active than the corresponding pentaosides. Peculiarities of the biosynthesis and biochemical evolution of glycosides of this type are discussed.
-
triterpene glycosides of sea cucumbers holothuroidea echinodermata as taxonomic markers
Natural Product Communications, 2015Co-Authors: Vladimir I Kalinin, Alexandra S Silchenko, Sergey A Avilov, Valentin A StonikAbstract:Triterpene glycosides are characteristic metabolites of sea cucumbers (Holothurioidea, Echinodermata). The majority of the glycosides belong to the holostane type {lanostane derivatives with an 18(20)-lactone}. Carbohydrate chains of these glycosides contain xylose, glucose, Quinovose, 3-O-methylglucose, and, rarely, 3-O-methylxylose, 3-O-methylglucuronic acid, 3-O-methylQuinovose, and 6-O-acetyl-glucose. The glycosides are specific for genera, groups of genera and even for species. The advantages and problems in the use of triterpene glycosides as taxonomic markers in the systematics of sea cucumbers are discussed.
-
immunomodulatory and anticancer activity of sea cucumber triterpene glycosides
Studies in natural products chemistry, 2014Co-Authors: Dmitry L Aminin, Evgeny A Pislyagin, Ekaterina S Menchinskaya, Alexandra S Silchenko, Sergey A Avilov, Vladimir I KalininAbstract:Abstract The triterpene glycosides are composed of a carbohydrate chain and triterpene aglycone and are widely distributed in sea cucumbers ( Holothurioidea , Echinodermata ). Most aglycones have 18(20)-lactones and belong to the holostane type. Carbohydrate chains of sea cucumber glycosides have from two to six monosaccharide residues including xylose, Quinovose, glucose, and 3- O -methylglucose and sometimes 3- O -methylxylose, 3- O -methylQuinovose, 3- O -methylglucuronic acid, and 6- O -acetylglucose. They may contain one, two, or three sulfate groups. At the milli- and micromolar concentrations, sea cucumber glycosides show hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. The basis of membranotropic action of the glycosides is their ability to attach to cell biomembranes and form nonselective ion-conducting complexes with 5(6)-nonsaturated sterol components of cell membranes, preferably with cholesterol. Such sterol/saponin interactions result in an efflux of some ions, nucleotides, and peptides, disrupting ion homeostasis and osmolarity followed by lysis and cell death. Some sea cucumber glycosides show an immunostimulatory effect at subtoxic nanomolar concentrations. Incubation of immune cells with the glycosides induces their activation resulting in an increase of immune cell adhesion on an extracellular matrix, enhancement of cell spreading and motility, increase of macrophage lysosomal activity, ROS formation, and phagocytic activity. The most effective immunostimulants are monosulfated glycosides, whereas di- and trisulfated glycosides are immunodepressants. Injection of subtoxic doses of some glycosides induces an increase in the number of antibody-producing plaque-forming cells in mouse spleens, an increase in the number, size and acidity of lysosomes of peritoneal macrophages, and increase of phagocytic index, resulting in heightened resistance by host animals against bacterial infections. Proteomic methods have demonstrated that the mechanism of immunomodulatory action of some sea cucumber glycosides on mouse splenocytes includes regulation of the expression of some proteins involved in the formation of cellular immune response. These glycosides regulate the expression of proteins associated with lysosome maturation, activation and merging, phagocytosis, cytoskeletal reorganization, cell adhesion, mobility, and proliferation of immune cells. It was shown that glycosides moderately induce production of some cytokines, restore the level of some CD markers of lymphocytes, increase bactericidal activity of leukocytes, and induce a significant increase in mouse resistance to lethal doses of some pathogenic microorganisms and radiation. Cytotoxic activity of sea cucumber glycosides against different types of cells and cell lines, including human tumor cell lines, has been studied for many years. These studies have shown the triterpene glycosides block egg cleavage and development of sea urchin embryos, suppress the proliferation of various human tumor cell lines in vitro , possess antiangiogenic effect, and cause cancer cell cycle arrest. Several sea cucumber glycosides are reported to induce tumor cell apoptosis in vitro and more importantly, IP administration in rodents of solutions of some sea cucumber triterpene glycosides show significant reduction of both tumor burden and metastasis. Recently, it was found that the new immunomodulatory lead compound, Cumaside, based on the holothurian triterpene glycoside, cucumarioside A 2 -2, demonstrates inhibition of tumor initiation and proliferation, in vivo and exhibits significant synergy with 5-fluorouracil.
Mark W Ruszczycky - One of the best experts on this subject based on the ideXlab platform.
-
epr kinetic isotope effect study of the mechanism of radical mediated dehydrogenation of an alcohol by the radical sam enzyme desii
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Mark W Ruszczycky, Sei Hyun ChoiAbstract:The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-Quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, , when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of , yielded a KIE of on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.
-
epr kinetic isotope effect study of the mechanism of radical mediated dehydrogenation of an alcohol by the radical sam enzyme desii
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Mark W Ruszczycky, Sei Hyun Choi, Hungwen LiuAbstract:The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-Quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, , when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of , yielded a KIE of on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.
-
stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical sam enzyme desii
Journal of the American Chemical Society, 2010Co-Authors: Mark W Ruszczycky, Sei Hyun Choi, Hungwen LiuAbstract:DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-Quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-d-glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 ± 0.05 and 1.01 ± 0.05 in the deamination and dehydrogenation reactions, respectively, using Na2S2O4 as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin ...
Sei Hyun Choi - One of the best experts on this subject based on the ideXlab platform.
-
epr kinetic isotope effect study of the mechanism of radical mediated dehydrogenation of an alcohol by the radical sam enzyme desii
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Mark W Ruszczycky, Sei Hyun ChoiAbstract:The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-Quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, , when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of , yielded a KIE of on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.
-
epr kinetic isotope effect study of the mechanism of radical mediated dehydrogenation of an alcohol by the radical sam enzyme desii
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Mark W Ruszczycky, Sei Hyun Choi, Hungwen LiuAbstract:The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-Quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, , when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of , yielded a KIE of on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.
-
stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical sam enzyme desii
Journal of the American Chemical Society, 2010Co-Authors: Mark W Ruszczycky, Sei Hyun Choi, Hungwen LiuAbstract:DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-Quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-d-glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 ± 0.05 and 1.01 ± 0.05 in the deamination and dehydrogenation reactions, respectively, using Na2S2O4 as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin ...
Hungwen Liu - One of the best experts on this subject based on the ideXlab platform.
-
epr kinetic isotope effect study of the mechanism of radical mediated dehydrogenation of an alcohol by the radical sam enzyme desii
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Mark W Ruszczycky, Sei Hyun Choi, Hungwen LiuAbstract:The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-Quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, , when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of , yielded a KIE of on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.
-
stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical sam enzyme desii
Journal of the American Chemical Society, 2010Co-Authors: Mark W Ruszczycky, Sei Hyun Choi, Hungwen LiuAbstract:DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-Quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-d-glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 ± 0.05 and 1.01 ± 0.05 in the deamination and dehydrogenation reactions, respectively, using Na2S2O4 as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin ...
