02009nas a2200205   4500008004100000022001400041245013400055210006900189260005700258300001200315490000700327520122100334100002701555700002301582700001801605700001601623700000901639700002701648856012801675       2013                        eng d  a0971-589400aMolecular characterization and differential expression studies of an oxidosqualene cyclase (OSC) gene of Brahmi (Bacopa monniera)0 aMolecular characterization and differential expression studies o  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcOCT  a547-5530 v193 a<p>Triterpenoid saponins are the class of secondary metabolites, synthesized via isoprenoid pathway. Oxidosqualene cyclases (OSCs) catalyzes the cyclization of 2, 3-oxidosqualene to various triterpene skeletons, the first committed step in triterpenoid biosynthesis. A full-length oxidosqualene cyclase cDNA from Bacopa monniera (BmOSC) was isolated and characterized. The open reading frame (ORF) of BmOSC consists of 2,292 bp, encoding 764 amino acid residues with an apparent molecular mass of 87.62 kDa and theoretical pI 6.21. It contained four QxxxxxW motifs, one Asp-Cys-Thr-Ala-Glu (DCTAE) motif which is highly conserved among the triterpene synthases and another MWCYCR motif involved in the formation of triterpenoid skeletons. The deduced amino acid sequence of BmOSC shares 80.5 % &amp; 71.8 % identity and 89.7 % &amp; 83.5 % similarity with Olea europaea mixed amyrin synthase and Panax notoginseng dammarenediol synthase respectively. Phylogenetic analysis revealed that BmOSC is closely related with other plant OSCs. Quantitative real-time PCR (qRT-PCR) data showed that BmOSC is expressed in all tissues examined with higher expression in stem and leaves as compared to roots and floral parts.</p>1 aVishwakarma, Rishi, K.1 aSonawane, Prashant1 aSingh, Somesh1 aKumari, Uma1 aRuby1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/molecular-characterization-and-differential-expression-studies-oxidosqualene-cyclase-osc02707nas a2200277   4500008004100000022001400041245015800055210006900213260005700282300001200339490000700351520166600358653003502024653002002059653002302079653002502102100002702127700000902154700001802163700002702181700002302208700001602231700002602247700002702273856012902300       2013                        eng d  a0735-964000aMolecular cloning, biochemical characterization, and differential expression of an acetyl-CoA C-acetyltransferase gene (AACT) of brahmi (Bacopa monniera)0 aMolecular cloning biochemical characterization and differential   a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcJUN  a547-5570 v313 a<p>Bacopa monniera (Brahmi) is an important Indian medicinal herb found in wet damp and marshy places. It produces medicinally important compounds known as bacosides along with alkaloids like brahmine and herpestine. Bacosides are triterpenoid saponins and their biosynthesis takes place via the isoprenoid pathway starting with acetyl-CoA. Acetyl-CoA C-acetyltransferase (AACT; EC 2.3.1.9), also known as acetoacetyl-CoA thiolase (Thiolase II), catalyzes the condensation of two acetyl-CoA to form 4-C compound acetoacetyl-CoA. Acetoacetyl-CoA is an important starting molecule for biosynthesis of various metabolites. Here, we report the cDNA cloning and characterization of acetyl-CoA C-acetyltransferase gene from B. monniera. The full-length gene was isolated using a RACE PCR protocol. The cDNA encoding AACT was designated as BmAACT (FJ947159) revealed an ORF of 1,218 bp and 405 amino acids, and shares 80 % similarity with other plant AACTs. Phylogenetic analysis showed that BmAACT is related closely to other dicot plants AACTs. The BmAACT gene was over-expressed in Escherichia coli as a 6X His-tag fusion protein and purified to homogeneity by Ni-NTA and gel filtration chromatography. Activity of recombinant protein was confirmed by thiolytic cleavage of acetoacetyl-CoA in the presence of 5 mM Mg2+, showing K (m) and V (max) of 20.67 mu M and 96.21 mu mol/min, respectively, with high catalytic efficiency (k (cat) = 2.30 x 10(5) min(-1)). Quantitative real-time PCR analysis showed that the expression of BmAACT is tissue-specific, and accumulation of transcripts is greater in roots and petals, followed by sepals, stem, leaf and pedicel.</p>10aAcetyl-CoA C-acetyltransferase10aBacopa monniera10aIsoprenoid pathway10aTriterpenoid saponin1 aVishwakarma, Rishi, K.1 aRuby1 aSingh, Somesh1 aSonawane, Prashant, D.1 aSrivastava, Sameer1 aKumari, Uma1 aKumar, R., J. Santosh1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/molecular-cloning-biochemical-characterization-and-differential-expression-acetyl-coa-c-002188nas a2200241   4500008004100000022001400041245009900055210006900154260007300223300001200296490000700308520130000315653004101615653002401656653002601680653001401706100002101720700002701741700001701768700001601785700002701801856011801828       2015                        eng d  a0141-813000aBacopa monniera recombinant mevalonate diphosphate decarboxylase: biochemical characterization0 aBacopa monniera recombinant mevalonate diphosphate decarboxylase  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcAUG  a661-6680 v793 a<p>Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg2+-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. K-m and V-max for MVAPP were 144 mu M and 52 U mg(-1) respectively. The values of turnover (k(cat)) and k(cat)/K-m, for mevalonate 5-diphosphate were determined to be 40 s(-1) and 2.77 x 10(5) M-1 s(-1) and k(cat) and k(cat)/K-m values for ATP were found to be 30 s(-1) and 2.20 x 10(4) M-1 s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 degrees C. The enzyme was most stable at pH 7 at 20 degrees C with the deactivation rate constant (K-d(*)) of 1.69 x 10(-4) and half life (t(1/2)) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg2+. (C) 2015 Elsevier B.V. All rights reserved.</p>10aMevalonate diphosphate decarboxylase10apH activity profile10aPhylogenetic analysis10astability1 aAbbassi, Shakeel1 aVishwakarma, Rishi, K.1 aPatel, Parth1 aKumari, Uma1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/bacopa-monniera-recombinant-mevalonate-diphosphate-decarboxylase-biochemical-102146nas a2200241   4500008004100000022001400041245008700055210006900142260007300211300001200284490000700296520129500303653002001598653002001618653002201638653001401660100001601674700002701690700002301717700002101740700002701761856011601788       2015                        eng d  a0141-813000aBiochemical characterization of recombinant mevalonate kinase from Bacopa monniera0 aBiochemical characterization of recombinant mevalonate kinase fr  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcJAN  a776-7830 v723 a<p>Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 degrees C, respectively. The enzyme was most stable at pH 8 at 25 degrees C with deactivation rate constant (Kd*) 1.398 x 10(-4) and half life (t(1/2)) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg2+, having K-m and V-max 331.9 mu M and 719.1 pKat mu g(-1), respectively. The values of k(cat) and k(cat)/K-m for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M-1 s(-1) and k(cat) and k(cat)/K-m values for ATP were found 150.9 s(-1) and 1.023 M-1 s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2. (C) 2014 Elsevier B.V. All rights reserved.</p>10aBacopa monniera10aEnzyme kinetics10aMevalonate kinase10astability1 aKumari, Uma1 aVishwakarma, Rishi, K.1 aSonawane, Prashant1 aAbbassi, Shakeel1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/biochemical-characterization-recombinant-mevalonate-kinase-bacopa-monniera-002468nas a2200325   4500008004100000022001400041245016100055210006900216260005700285300001400342490000700356520130200363653001901665653002001684653003701704653002801741653002301769653002201792100002701814700001801841700002301859700001601882700001801898700000901916700002101925700002401946700002101970700002701991856012402018       2015                        eng d  a0735-964000ac Squalene synthase gene from medicinal herb bacopa monniera: molecular characterization, differential expression, comparative modeling, and docking studies0 ac Squalene synthase gene from medicinal herb bacopa monniera mol  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcDEC  a1675-16850 v333 a<p>The widespread pharmaceutically important triterpenoid saponins are synthesized via isoprenoid pathway. The formation of squalene is the key regulatory point in triterpene biosynthesis, catalyzed by squalene synthase (SQS). The present study deals with the detailed characterization of SQS by molecular, biochemical, and computational means from Bacopa monniera, an immensely important medicinal plant rich in triterpenoid saponin, bacosides. A full-length SQS gene was isolated from B. monniera, characterized as B. monniera squalene synthase (BmSQS) (1242 bp) encoding 414 amino acids. Deduced amino acid sequence of BmSQS showed highly conserved consensus aspartate-rich motifs (DXXXD) and catalytic site residues. Phylogenetic analysis showed that BmSQS belongs to dicot group having closest relationship with Salvia miltiorrhiza. Semiquantitative and real-time PCR studies showed that the BmSQS messenger RNA (mRNA) expression level was higher in vegetative parts (roots) as compared to floral parts. Methyl jasmonate induces the BmSQS mRNA expression in all tissues tested, while salicylic acid, cold, and salt induce much higher expression in roots. Homology modeling and docking simulations of BmSQS showed the pivotal roles of Asp77, Asp81, Asp213, Asp217, and Tyr168 in catalysis.</p>10aAbiotic stress10aBacopa monniera10aComparative modeling and docking10aDifferential expression10aIsoprenoid pathway10aSqualene synthase1 aVishwakarma, Rishi, K.1 aPatel, Krunal1 aSonawane, Prashant1 aKumari, Uma1 aSingh, Somesh1 aRuby1 aAbbassi, Shakeel1 aAgrawal, Dinesh, C.1 aTsay, Hsin-Sheng1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/c-squalene-synthase-gene-medicinal-herb-bacopa-monniera-molecular-characterization-002140nas a2200205   4500008004100000022001400041245008000055210006900135260005700204300001200261490000700273520142200280100001601702700002701718700001601745700000901761700002301770700002701793856011401820       2015                        eng d  a0971-589400aEfficient shoots regeneration and genetic transformation of bacopa monniera0 aEfficient shoots regeneration and genetic transformation of baco  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcAPR  a261-2670 v213 a<p>Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles. Present report describes efficient in vitro multiplication and transformation method for geneticmanipulation of this species. MS medium supplemented with 2 mgl(-1) BA and 0.2 mgl(-1) IAA was found optimum for maximum shoot regeneration (98.33 %) from in vitro leaves with 2-3 longitudinal cuts. Agrobacterium tumefaciens-mediated transformation method was used for generating transgenic B. monniera plants. Putative transformants were confirmed by GUS assay and PCR based confirmation of hptII gene. DNA blot analysis showed single copy insertion of transgene cassette. An average of 87.5 % of the regenerated shoots were found PCR positive for hptII gene and GUS activity was detected in leaves of transgenic shoots at a frequency of 82.5 % The efficient multiple shoots regeneration system described herein may help in mass production of B. monniera plant. Also, the high frequency transformation protocol described here can be used for genetic engineering of B. monniera for enhancement of its pharmaceutically important metabolites.</p>1 aKumari, Uma1 aVishwakarma, Rishi, K.1 aGupta, Neha1 aRuby1 aShirgurkar, M., V.1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/efficient-shoots-regeneration-and-genetic-transformation-bacopa-monniera-0