02593nas a2200265   4500008004100000022001400041245014100055210006900196260010300265300001200368490000700380520154900387653002801936653002401964653002601988653002402014100002302038700002502061700001702086700002702103700002202130700002402152700002702176856012402203       2011                        eng d  a0981-942800aExpression analysis of cinnamoyl-CoA reductase (CCR) gene in developing seedlings of Leucaena leucocephala: A pulp yielding tree species0 aExpression analysis of cinnamoylCoA reductase CCR gene in develo  a23 RUE LINOIS, 75724 PARIS, FRANCEbELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIERcFEB  a138-1450 v493 a<p>Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as LI-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that LI-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L leucocephala contains two copies as revealed by DNA blot hybridization. LI-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that LI-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PcR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5,10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry. (C) 2010 Elsevier Masson SAS. All rights reserved.</p>10aCinnamoyl-CoA reductase10aDeveloping seedling10aLeucaena leucocephala10aLignin biosynthesis1 aSrivastava, Sameer1 aGupta, Ranadheer, K.1 aArha, Manish1 aVishwakarma, Rishi, K.1 aRawal, Shuban, K.1 aKishor, P., B. Kavi1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/expression-analysis-cinnamoyl-coa-reductase-ccr-gene-developing-seedlings-leucaena-002378nas a2200241   4500008004100000022001400041245011300055210006900168260007400237300001400311490000700325520145200332653004801784653001201832653002201844653002301866100001601889700001901905700002601924700002701950700002701977856013202004       2012                        eng d  a0301-485100aFunctional characterization and differential expression studies of squalene synthase from Withania somnifera0 aFunctional characterization and differential expression studies   aVAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDSbSPRINGERcSEP  a8803-88120 v393 a<p>Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His-Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni-NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph-mass spectrometer (GC-MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.</p>10aGas chromatograph-Mass Spectrometer (GC-MS)10aqRT-PCR10aSqualene synthase10aWithania somnifera1 aGupta, Neha1 aSharma, Poonam1 aKumar, R., J. Santosh1 aVishwakarma, Rishi, K.1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/functional-characterization-and-differential-expression-studies-squalene-synthase-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<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-001717nas a2200265   4500008004100000022001400041245008300055210006900138260007300207300001200280490000800292520084600300653001701146653001301163653001801176653001601194653001401210653000801224100001301232700002701245700002701272700002101299700001701320856011401337       2014                        eng d  a0167-577X00aBiological synthesis of cationic gold nanoparticles and binding of plasmid DNA0 aBiological synthesis of cationic gold nanoparticles and binding   aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcAUG  a159-1610 v1293 a<p>Nanobiotechnology is the development of eco-friendly experimental processes for the synthesis of nanomaterials. The present work focuses on synthesis of cationic gold nanoparticles (C-GNPs) for biological applications, especially in gene and drug delivery studies. A biosynthesis methodology has been developed for the functionalization of gold nanoparticles to cationic nature. The synthesis of C-GNPs was done by using peanut leaf extract in the presence of cysteamine. The formed C-GNPs were characterized by using UV-visible spectroscopy (UV-vis), the particles sizes and shapes were confirmed by a Transmission electron microscope (TEM) and crystallinity of C-GNPs was characterized by diffraction. The binding of plasmid DNA on the C-GNPs was confirmed by agarose gel electrophoresis. (C) 2014 Elsevier B.V. All rights reserved.</p>10aBiomaterials10aCationic10aNanoparticles10aPlasmid DNA10asynthesis10aTEM1 aRaju, D.1 aVishwakarma, Rishi, K.1 aKhan, Bashir, Mohammad1 aMehta, Urmil, J.1 aAhmad, Absar  uhttps://library.ncl.res.in/content/biological-synthesis-cationic-gold-nanoparticles-and-binding-plasmid-dna-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-002316nas a2200193   4500008004100000022001400041245015600055210006900211260005700280300001200337490000700349520151600356100002301872700002701895700002301922700002201945700002701967856012801994       2015                        eng d  a0971-589400aAbiotic stress induces change in cinnamoyl CoA reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala0 aAbiotic stress induces change in cinnamoyl CoA reductase CCR pro  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcAPR  a197-2050 v213 a<p>Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.</p>1 aSrivastava, Sameer1 aVishwakarma, Rishi, K.1 aArafat, Yasir, Ali1 aGupta, Sushim, K.1 aKhan, Bashir, Mohammad  uhttps://library.ncl.res.in/content/abiotic-stress-induces-change-cinnamoyl-coa-reductase-ccr-protein-abundance-and-lignin-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-002232nas a2200181   4500008004100000022001500041245010100056210006900157260000800226490000700234520156800241100001801809700002201827700002701849700002701876700002101903856012601924       2018                        eng d  a 0959-399300a Enhanced activity of Withania somnifera family-1 glycosyltransferase (UGT73A16) via mutagenesis0 aEnhanced activity of Withania somnifera family1 glycosyltransfer  cOCT0 v343 aThis work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein-ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2-3 fold and 6-7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.1 aSingh, Somesh1 aPatel, Krunal, A.1 aSonawane, Prashant, D.1 aVishwakarma, Rishi, K.1 aKhan, Bashir, M.  uhttps://library.ncl.res.in/content/enhanced-activity-withania-somnifera-family-1-glycosyltransferase-ugt73a16-mutagenesis