00625nas a2200157 4500008004100000245013100041210006900172260000800241300001400249490000700263100001700270700000900287700002700296700001700323856012700340 2011 eng d00aSilico substrate specificity in bmgt1 and bmgt2 genes of bacopa monniera glycosyltransferases online journal of bioinformatics0 aSilico substrate specificity in bmgt1 and bmgt2 genes of bacopa  cJAN a413 - 4300 v121 aSharma, Ranu1 aRuby1 aKhan, Bashir, Mohammad1 aSuresh, C.G. uhttps://library.ncl.res.in/content/silico-substrate-specificity-bmgt1-and-bmgt2-genes-bacopa-monniera-glycosyltransferases02540nas a2200277 4500008004100000022001400041245011000055210006900165260007900234300001200313490000800325520151400333653001401847653001801861653002401879653002301903653005501926653002301981100001802004700002802022700002602050700002702076700000902103700002702112856012302139 2013 eng d a0273-228900aFunctional characterization of a flavonoid glycosyltransferase gene from withania somnifera (Ashwagandha)0 aFunctional characterization of a flavonoid glycosyltransferase g a999 RIVERVIEW DRIVE SUITE 208, TOTOWA, NJ 07512 USAbHUMANA PRESS INCcJUN a729-7410 v1703 a

Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W. somnifera (UGT73A16) showing 85-92 % homology with UGTs from other plants. UGT73A16 was expressed as a His(6)-tagged fusion protein in Escherichia coli. Several compounds, including flavonoids, were screened as potential substrates for UGT73A16. HPLC analysis and hypsochromic shift indicated that UGT73A16 transfers a glucose molecule to several different flavonoids. Based on kinetic parameters, UGT73A16 shows more catalytic efficiency towards naringenin. Here, we explored UGT73A16 of W. somnifera as whole cell catalyst in E. coli. We used flavonoids (genistein, apigenin, kaempferol, naringenin, biochanin A, and daidzein) as substrates for this study. More than 95 % of the glucoside products were released into the medium, facilitating their isolation. Glycosylation of substrates occurred on the 7- and 3-hydroxyl group of the aglycone. UGT73A16 also displayed regiospecific glucosyl transfer activity towards 3-hydroxy flavone compound, which is the backbone of all flavonols and also for a chemically synthesized compound, not found naturally. The present study generates essential knowledge and molecular as well as biochemical tools that allow the verification of UGT73A16 in glycosylation.

10aFlavonoid10aglycosylation10aGlycosyltransferase10aHypsochromic shift10aPlant secondary product glycosyltransferase (PSPG)10aWithania somnifera1 aSingh, Somesh1 aVishwakarma, Rishi., K.1 aKumar, R., J. Santosh1 aSonawane, Prashant, D.1 aRuby1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/functional-characterization-flavonoid-glycosyltransferase-gene-withania-somnifera-002294nas a2200253 4500008004100000022001400041245011800055210006900173260005700242300001400299490000700313520138200320653001301702653001501715653002401730653002301754100002601777700000901803700001801812700002701830700002401857700002701881856013201908 2013 eng d a0735-964000aFunctional characterization of a glucosyltransferase specific to flavonoid 7-O-glucosides from withania somnifera0 aFunctional characterization of a glucosyltransferase specific to a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcOCT a1100-11080 v313 a

Flavonoids are a large class of phenylpropanoid-derived secondary metabolites, which are usually glycosylated by UDP-glycosyltransferases with one or more sugar groups. Here, we report the cloning and biochemical characterization of a flavonoid glycosyltransferase gene from Withania somnifera (WsGT), which is an important medicinal plant used in Ayurvedic formulations. Using PCR primers, designed for a highly conserved region of previously reported glycosyltransferases, we were able to isolate the corresponding fragment of the WsGT gene. Rapid amplification of cDNA ends (RACE) was then employed to isolate full-length cDNA, which had an open reading frame of 1,371 bp that encode for 456 amino acids. Phylogenetic analysis indicated that WsGT was similar to that of family 1 GT-B glycosyltransferase. Biochemical analysis revealed that WsGT interacts with UDP-glucose and was capable of regiospecifically glycosylating flavonoid-7-ols, such as apigenin, naringenin, luteolin, diadzein and genistein. Expression profiling studies showed that WsGT was highly expressed in young and mature leaves of W. somnifera. Furthermore, exposure to salicylic acid enhanced the expression of WsGT in the leaves and heat shock treatment resulted in decreased expression of WsGT after an initial increase. This may suggest the role of WsGT in response to abiotic/biotic stresses.

10aDiadzein10aFlavonoids10aGlycosyltransferase10aWithania somnifera1 aKumar, R., J. Santosh1 aRuby1 aSingh, Somesh1 aSonawane, Prashant, D.1 aVishwakarma, R., K.1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/functional-characterization-glucosyltransferase-specific-flavonoid-7-o-glucosides-withania-002009nas 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

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 % & 71.8 % identity and 89.7 % & 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.

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

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.

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

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.

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-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

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.

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

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.

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