02707nas 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-002607nas a2200253   4500008004100000022001400041245012100055210006900176260007400245300001400319490000700333520164700340653002001987653002402007653002402031653002402055653005402079100000902133700002602142700002702168700001802195700002702213856011302240       2014                        eng d  a0301-485100aMolecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera0 aMolecular cloning and characterization of genistein 4Oglucoside   aVAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDSbSPRINGERcJUL  a4675-46880 v413 a<p>Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49 % identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.</p>10aBacopa monniera10aExpression analysis10aGlycosyltransferase10aImmuno-localization10aPlant secondary product glycosyltransferase motif1 aRuby1 aKumar, R., J. Santosh1 aVishwakarma, Rishi, K.1 aSingh, Somesh1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/molecular-cloning-and-characterization-genistein-4-o-glucoside-specific-002146nas 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-0