@article {42770, title = {Silico substrate specificity in bmgt1 and bmgt2 genes of bacopa monniera glycosyltransferases online journal of bioinformatics}, journal = {Bioinformatics}, volume = {12}, year = {2011}, month = {JAN}, pages = {413 - 430}, author = {Sharma, Ranu and Ruby and Khan, Bashir Mohammad and Suresh, C.G.} } @article { ISI:000319134800020, title = {Functional characterization of a flavonoid glycosyltransferase gene from withania somnifera (Ashwagandha)}, journal = {Applied Biochemistry and Biotechnology}, volume = {170}, number = {3}, year = {2013}, month = {JUN}, pages = {729-741}, publisher = {HUMANA PRESS INC}, address = {999 RIVERVIEW DRIVE SUITE 208, TOTOWA, NJ 07512 USA}, abstract = {

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.

}, keywords = {Flavonoid, glycosylation, Glycosyltransferase, Hypsochromic shift, Plant secondary product glycosyltransferase (PSPG), Withania somnifera}, issn = {0273-2289}, doi = {10.1007/s12010-013-0230-2}, author = {Singh, Somesh and Vishwakarma, Rishi. K. and Kumar, R. J. Santosh and Sonawane, Prashant D. and Ruby and Khan, Bashir Mohammad} } @article { ISI:000323669900005, title = {Functional characterization of a glucosyltransferase specific to flavonoid 7-O-glucosides from withania somnifera}, journal = {Plant Molecular Biology Reporter}, volume = {31}, number = {5}, year = {2013}, month = {OCT}, pages = {1100-1108}, publisher = {SPRINGER}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

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.

}, keywords = {Diadzein, Flavonoids, Glycosyltransferase, Withania somnifera}, issn = {0735-9640}, doi = {10.1007/s11105-013-0573-4}, author = {Kumar, R. J. Santosh and Ruby and Singh, Somesh and Sonawane, Prashant D. and Vishwakarma, R. K. and Khan, Bashir Mohammad} } @article { ISI:000324794900010, title = {Molecular characterization and differential expression studies of an oxidosqualene cyclase (OSC) gene of Brahmi (Bacopa monniera)}, journal = {Physiology and Molecular Biology of Plants}, volume = {19}, number = {4}, year = {2013}, month = {OCT}, pages = {547-553}, publisher = {SPRINGER}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

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.

}, issn = {0971-5894}, doi = {10.1007/s12298-013-0195-1}, author = {Vishwakarma, Rishi K. and Sonawane, Prashant and Singh, Somesh and Kumari, Uma and Ruby and Khan, Bashir Mohammad} } @article { ISI:000319073500006, title = {Molecular cloning, biochemical characterization, and differential expression of an acetyl-CoA C-acetyltransferase gene (AACT) of brahmi (Bacopa monniera)}, journal = {Plant Molecular Biology Reporter}, volume = {31}, number = {3}, year = {2013}, month = {JUN}, pages = {547-557}, publisher = {SPRINGER}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

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.

}, keywords = {Acetyl-CoA C-acetyltransferase, Bacopa monniera, Isoprenoid pathway, Triterpenoid saponin}, issn = {0735-9640}, doi = {10.1007/s11105-012-0523-6}, author = {Vishwakarma, Rishi K. and Ruby and Singh, Somesh and Sonawane, Prashant D. and Srivastava, Sameer and Kumari, Uma and Kumar, R. J. Santosh and Khan, Bashir Mohammad} } @article { ISI:000338283000052, title = {Molecular cloning and characterization of genistein 4{\textquoteright}-O-glucoside specific glycosyltransferase from Bacopa monniera}, journal = {Molecular Biology Reports}, volume = {41}, number = {7}, year = {2014}, month = {JUL}, pages = {4675-4688}, publisher = {SPRINGER}, address = {VAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDS}, abstract = {

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{\textquoteright}-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4{\textquoteright}-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{\textquoteright}-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.

}, keywords = {Bacopa monniera, Expression analysis, Glycosyltransferase, Immuno-localization, Plant secondary product glycosyltransferase motif}, issn = {0301-4851}, doi = {10.1007/s11033-014-3338-8}, author = {Ruby and Kumar, R. J. Santosh and Vishwakarma, Rishi K. and Singh, Somesh and Khan, Bashir Mohammad} } @article { ISI:000367464000006, title = {c Squalene synthase gene from medicinal herb bacopa monniera: molecular characterization, differential expression, comparative modeling, and docking studies}, journal = {Plant Molecular Biology Reporter}, volume = {33}, number = {6}, year = {2015}, month = {DEC}, pages = {1675-1685}, publisher = {SPRINGER}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

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.

}, keywords = {Abiotic stress, Bacopa monniera, Comparative modeling and docking, Differential expression, Isoprenoid pathway, Squalene synthase}, issn = {0735-9640}, doi = {10.1007/s11105-015-0864-z}, author = {Vishwakarma, Rishi K. and Patel, Krunal and Sonawane, Prashant and Kumari, Uma and Singh, Somesh and Ruby and Shakeel Abbassi and Agrawal, Dinesh C. and Tsay, Hsin-Sheng and Khan, Bashir Mohammad} } @article { ISI:000357587500011, title = {Efficient shoots regeneration and genetic transformation of bacopa monniera}, journal = {Physiology and Molecular Biology of Plants}, volume = {21}, number = {2}, year = {2015}, month = {APR}, pages = {261-267}, publisher = {SPRINGER}, type = {Article}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

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.

}, issn = {0971-5894}, doi = {10.1007/s12298-015-0290-6}, author = {Kumari, Uma and Vishwakarma, Rishi K. and Gupta, Neha and Ruby and Shirgurkar, M. V. and Khan, Bashir Mohammad} }