02875nas a2200349 4500008004100000022001400041245015400055210006900209260011000278300001200388490000600400520159700406653002302003653002502026653002902051653002402080100002002104700001702124700001802141700001802159700001802177700001702195700001802212700002402230700001802254700002102272700003002293700002702323700002202350700002402372856012902396 2008 eng d a0972-584900aDirect shoot organogenesis and plant regeneration from hypocotyl explants in selected genotypes of Leucaena leucocephala - a leguminous pulpwood tree0 aDirect shoot organogenesis and plant regeneration from hypocotyl aDR K S KRISHNAN MARG, PUSA CAMPUS, NEW DELHI 110 012, INDIAbNATL INST SCIENCE COMMUNICATION-NISCAIRcJUL a388-3930 v73 a

An efficient in vitro plant regeneration system in subabul (Leucaena leucocephala), a leguminous pulp wood tree species, was established. The induction of shoots was achieved from selected elite clones of subabul K-8, K-636 and also wild type on MS medium supplemented with 2% sucrose and different concentrations (0.88 to 24.6 mu M) of plant growth regulators (BA, Kn, 2iP & TDZ). The best medium for shoot regeneration was MS with 22.2 mu M BA (5 shoots per explant), followed by 22.7 mu M TDZ (4.6 shoots per explant). Addition of putriscine (9.3 mu M) to MS medium containing 22.2 mu M BA enhanced the number of multiple shoots to 7-8 but not the frequency of response. Shoot initials (measuring 1 cm) when separated and transferred on to MS medium containing 1.4 mu M GA(3) elongated to 2-5 cm in 15.20 d with 80% frequency. The per cent frequency of shoot differentiation was almost identical in the genotypes K-8 and K-636 but it differed significantly from the wild type. Leaf yellowing and abscission in all the genotypes was curtailed by supplementing the medium with 685 mu M glutamine or 540 mu M adenine. The excised shoots were transferred to root regeneration media containing 2.46 and 4.98 mu M IBA or 2.6 and 5.3 mu M NAA. Root regeneration was noticed with 100% frequency in all the three genotypes in presence of IBA or NAA. Plantlets were transferred successfully to the pots with 70% survival rate with no visible morphological variations. The protocol can be utilized for mass propagation and genetic transformation studies of this important pulpwood species.

10ahypocotyl explants10aLeucaena lucocephala10apulpwood leguminous tree10ashoot organogenesis1 aSirisha, V., L.1 aPrashant, S.1 aRanadheer, D.1 aRamprasad, P.1 aShaik, N., M.1 aArha, Manish1 aGupta, S., K.1 aSrivastava, Sarneer1 aYadav, A., K.1 aKulkarni, P., S.1 aAbhilash, Othalathara, U.1 aKhan, Bashir, Mohammad1 aRawal, Shuban, K.1 aKishor, P., B. Kavi uhttps://library.ncl.res.in/content/direct-shoot-organogenesis-and-plant-regeneration-hypocotyl-explants-selected-genotypes-002067nas a2200229 4500008004100000022001400041245009900055210006900154260005700223300001400280490000700294520120800301653003101509653003001540653001701570653002501587100002401612700002401636700002701660700002201687856012801709 2008 eng d a0959-399300aIsolation and heterologous expression of PHA synthesising genes from Bacillus thuringiensis R10 aIsolation and heterologous expression of PHA synthesising genes  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcSEP a1769-17740 v243 a

The polyhydroxyalkanoate biosynthesis gene locus from Bacillus thuringiensis R1 was isolated, cloned and analyzed at the molecular level. We found that a similar to 5 kb SacI-ClaI digested fragment of genomic DNA from B. thuringiensis R1 encoding the PHA synthesising genes, conferred PHA producing ability to E. coli. The fragment was sequenced and found to be of 4787 bp with five open reading frames. Sequence alignment with closely related species of Bacillus in the existing database revealed that the ORFs correspond to phaP, phaQ, phaR, phaB and phaC genes. However, E. coli harboring phaP, phaQ, phaR, phaB and phaC locus produced very low PHA. Furthermore, complementation of the locus with phaA from Ralstonia eutropha increased the PHA production in the recombinant E. coli from 3.0% to 24% of cell dry mass. The putative promoter regions and ribosome binding sites were identified for each of the gene. Conserved domains for PHA synthase and aceto-acetyl-coA reductase were also identified. We hence conclude that the PHA operon of Bacillus thuringiensis R1 consists of phaP, phaQ, phaR, phaB, phaC and complementation of the same with phaA is accountable for its high PHA production.

10aaceto-acetyl-coA reductase10aBacillus thuringiensis R110aPHA synthase10apolyhydroxyalkanoate1 aDesetty, Rohini, D.1 aMahajan, Vineet, S.1 aKhan, Bashir, Mohammad1 aRawal, Shuban, K. uhttps://library.ncl.res.in/content/isolation-and-heterologous-expression-pha-synthesising-genes-bacillus-thuringiensis-r1-002176nas a2200241 4500008004100000022001400041245010200055210006900157260010700226300001200333490000700345520124900352653002401601653002201625653003301647653001701680653001601697100001601713700002301729700002701752700003101779856012401810 2009 eng d a1431-065100aCharacterization of the smallest dimeric bile salt hydrolase from a thermophile brevibacillus sp.0 aCharacterization of the smallest dimeric bile salt hydrolase fro aCHIYODA FIRST BLDG EAST, 3-8-1 NISHI-KANDA, CHIYODA-KU, TOKYO, 101-0065, JAPANbSPRINGER JAPAN KKcMAR a363-3700 v133 a

A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India. This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis were 9.0 and 60A degrees C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K (M) and k (cat) values of 3.08 mu M and 6.32 x 10(2) s(-1), respectively, for glycodeoxycholic acid. The NH2-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases. To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source.

10aBile salt hydrolase10aBrevibacillus sp.10aDimeric intracellular enzyme10aPurification10aThermophile1 aSridevi, N.1 aSrivastava, Sameer1 aKhan, Bashir, Mohammad1 aPrabhune, Asmita, Ashutosh uhttps://library.ncl.res.in/content/characterization-smallest-dimeric-bile-salt-hydrolase-thermophile-brevibacillus-sp-003067nas a2200301 4500008004100000022001400041245019700055210006900252260005700321300001200378490000700390520193100397653004002328653001202368653001402380653002802394100003002422700001702452700001802469700002102487700002002508700001702528700002102545700002702566700001802593700002402611856013002635 2009 eng d a1610-294000aPhylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl-CoA-O- methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala)0 aPhylogenetic analysis homology modelling molecular dynamics and  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcFEB a203-2210 v153 a

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 alpha-helices, 6 and 7 beta-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.

10aCaffeoyl-CoA 3-O-methyl transferase10adocking10aModelling10aS-adenosyl homocysteine1 aPagadala, Nataraj, Sekhar1 aArha, Manish1 aReddy, P., S.1 aKumar, Ranadheer1 aSirisha, V., L.1 aPrashant, S.1 aReddy, Janardhan1 aKhan, Bashir, Mohammad1 aRawal, S., K.1 aKishor, P., B. Kavi uhttps://library.ncl.res.in/content/phylogenetic-analysis-homology-modelling-molecular-dynamics-and-docking-studies-caffeoyl-001704nas a2200181 4500008004100000022001400041245007000055210006700125260007700192300001400269490000700283520100900290100003201299700003001331700002701361700003001388856010401418 2010 eng d a1616-301X00aNanogold-Loaded sharp-edged carbon bullets as plant-gene carriers0 aNanogoldLoaded sharpedged carbon bullets as plantgene carriers aPO BOX 10 11 61, D-69451 WEINHEIM, GERMANYbWILEY-V C H VERLAG GMBHcAUG a2416-24230 v203 a

The higher DNA delivery efficiency into plants by gold nanoparticles embedded in sharp carbonaceous carriers is demonstrated. These nanogold-embedded carbon matrices are prepared by heat treatment of biogenic intracellular gold nanoparticles. The DNA-delivery efficiency is tested on a model plant, Nicotiana tabacum, and is further extended to the monocot, Oryza sativa, and a hard dicot tree species, Leucaena leucocephala. These materials reveal good dispersion of the transport material, producing a greater number of GUS foci per unit area. The added advantages of the composite carrier are the lower plasmid and gold requirements. Plant-cell damage with the carbon-supported particles is very minimal and can be, gauged from the increased plant regeneration and transformation efficiency compared with that of the commercial micrometer-sized gold particles. This is ascribed to the sharp edges that the carbon supports possess, which lead to better piercing capabilities with minimum damage.

1 aVijayakumar, Periyasamy, S.1 aAbhilash, Othalathara, U.1 aKhan, Bashir, Mohammad1 aPrasad, Bhagavatula L. V. uhttps://library.ncl.res.in/content/nanogold-loaded-sharp-edged-carbon-bullets-plant-gene-carriers-002593nas 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

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.

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-000625nas 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-glycosyltransferases02944nas a2200325 4500008004100000022001400041245017600055210006900231260007400300300001200374490000700386520175900393653003502152653002702187653003602214653001202250100002002262700001702282700002102299700001502320700001502335700001702350700002002367700001902387700001902406700002002425700002702445700002402472856012202496 2012 eng d a0167-690300aCloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco0 aCloning characterization and impact of up and downregulating sub aVAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDSbSPRINGERcAPR a239-2530 v663 a

Both cDNA including 5'UTR and 3'UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium x Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5'UTR and 283 bases of 3'UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7-32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in up-regulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.

10aCinnamyl alcohol dehydrogenase10aLignin down-regulation10aSubabul (Leucaena leucocephala)10aTobacco1 aSirisha, V., L.1 aPrashant, S.1 aKumar, Ranadheer1 aPramod, S.1 aJalaja, N.1 aKumari, Hima1 aRao, Maheshwari1 aRao, Nageswara1 aMishra, Preeti1 aKarumanchi, Rao1 aKhan, Bashir, Mohammad1 aKishor, P., B. Kavi uhttps://library.ncl.res.in/content/cloning-characterization-and-impact-and-down-regulating-subabul-cinnamyl-alcohol-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

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.

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-001978nas a2200229 4500008004100000022001400041245011200055210006900167260007300236300001200309490000700321520111200328653002201440653002501462653003001487653000901517653001401526100002301540700003201563700002701595856012601622 2013 eng d a0141-813000aBiochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala0 aBiochemical characterization of recombinant cinnamoyl CoA reduct aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcJUL a154-1590 v583 a

Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25 degrees C for 90 min. The enzyme showed k(cat)/k(m) for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (x10(6) M-1 s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, E-a for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme. (C) 2013 Elsevier B.V. All rights reserved.

10aActivation energy10aCinnamoyl CoA esters10aCinnamoyl CoA reductase 110aSAXS10astability1 aSonawane, Prashant1 aVishwakarma, Rishi, Kishore1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/biochemical-characterization-recombinant-cinnamoyl-coa-reductase-1-ll-ccrh1-leucaena-002168nas a2200229 4500008004100000022001400041245010000055210006900155260007400224300001400298490000700312520132000319653003401639653002601673653001101699653002001710100001901730700001701749700001801766700002701784856012701811 2013 eng d a0301-485100aCinnamate 4-hydroxylase (C4H) genes from leucaena leucocephala: a pulp yielding leguminous tree0 aCinnamate 4hydroxylase C4H genes from leucaena leucocephala a pu aVAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDSbSPRINGERcFEB a1265-12740 v403 a

Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 1.14.13.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age.

10aCinnamate 4-Hydroxylase (C4H)10aLeucaena leucocephala10alignin10aPhenylpropanoid1 aKumar, Santosh1 aOmer, Sumita1 aPatel, Krunal1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/cinnamate-4-hydroxylase-c4h-genes-leucaena-leucocephala-pulp-yielding-leguminous-tree-002628nas a2200265 4500008004100000022001400041245019800055210006900253260007400322300001400396490000700410520155800417653003201975653002202007653002602029653002202055100002002077700001802097700001802115700002402133700003202157700002202189700002702211856012402238 2013 eng d a0301-485100aFunctional characterization, homology modeling and docking studies of beta-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)0 aFunctional characterization homology modeling and docking studie aVAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDSbSPRINGERcFEB a1351-13630 v403 a

Glycosyl hydrolase family 1 beta-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving beta-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this beta-glucosidase were found to be 45 A degrees C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K (m) and V (max) were found to be 38.59 mu M and 0.8237 mu M/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 mu M and 0.1037 mu M/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (Delta G) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.

10aGlycosyl hydrolase family 110ahomology modeling10aLeucaena leucocephala10aMolecular docking1 aShaik, Noor, M.1 aMisra, Anurag1 aSingh, Somesh1 aFatangare, Amol, B.1 aRamakumar, Suryanarayanarao1 aRawal, Shuban, K.1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/functional-characterization-homology-modeling-and-docking-studies-beta-glucosidase-002540nas 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-002411nas a2200229 4500008004100000022001400041245014800055210006900203260005700272300001200329490000700341520155600348653002601904653001101930653001201941653001401953653002101967100001701988700001902005700002702024856013002051 2013 eng d a0721-771400aOver-expression of a subgroup 4 R2R3 type MYB transcription factor gene from Leucaena leucocephala reduces lignin content in transgenic tobacco0 aOverexpression of a subgroup 4 R2R3 type MYB transcription facto a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcJAN a161-1710 v323 a

LlMYB1 , a subgroup 4 R2R3-type MYB transcription factor gene from Leucaena leucocephala appears to be a repressor of lignin biosynthesis pathway by regulating the transcription of general phenylpropanoid pathway genes. R2R3MYB transcription factors are known to play a wide role in regulating the phenylpropanoid pathway in plants. In this study, we report isolation, cloning and characterization of an R2R3MYB transcription factor gene (LlMYB1) from an economically important tree species, Leucaena leucocephala. LlMYB1 consists of 705 bp coding sequence corresponding to 235 amino acids. Sequence alignment revealed that the N-terminal (MYB) domain of the gene shares up to 95 % similarity with subgroup 4 (Sg4) members of R2R3Myb gene family functionally known to be lignin repressors. Highly divergent C-terminal region of the gene carried an ERF-associated amphiphilic repression (EAR) motif, another characteristic of the Sg4. The gene was phylogenetically grouped closest with AmMYB308, a known repressor of monolignol biosynthetic pathway genes. Spatio-temporal expression studies at different ages of seedlings using quantitative real-time PCR (QRT-PCR) showed highest transcript level of the gene in 10 day old stem tissues. Over-expression of the gene in transgenic tobacco showed statistically significant decline in the transcript levels of the general phenylpropanoid pathway genes and reduction in lignin content. Our study suggests that LlMYB1 might be playing the role of a repressor of lignin biosynthesis in L. leucocephala.

10aLeucaena leucocephala10alignin10aR2R3MYB10aRepressor10aTranscript level1 aOmer, Sumita1 aKumar, Santosh1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/over-expression-subgroup-4-r2r3-type-myb-transcription-factor-gene-leucaena-leucocephala-002183nas a2200289 4500008004100000022001400041245009500055210006900150260007300219300001000292490000700302520114300309653002601452653003001478653002401508653002201532653003001554653002501584100002301609700001801632700003201650700002301682700001801705700002501723700002701748856011801775 2013 eng d a0141-813000aProbing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala0 aProbing the active site of cinnamoyl CoA reductase 1 LlCCRH1 fro aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcSEP a33-380 v603 a

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in K-m values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1. (c) 2013 Elsevier B.V. All rights reserved.

10aChemical modification10aCinnamoyl CoA reductase 110aDocking simulations10ahomology modeling10aSite directed mutagenesis10aSubstrate protection1 aSonawane, Prashant1 aPatel, Krunal1 aVishwakarma, Rishi, Kishore1 aSrivastava, Sameer1 aSingh, Somesh1 aGaikwad, Sushama, M.1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/probing-active-site-cinnamoyl-coa-reductase-1-ll-ccrh1-leucaena-leucocephala-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

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.

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-002079nas a2200229 4500008004100000022001400041245008700055210006900142260007300211300001000284490000700294520124300301653004001544653001701584653001901601653002001620653001401640100002701654700002701681700002501708856011601733 2014 eng d a0141-813000aConformational transitions of cinnamoyl CoA reductase 1 from leucaena leucocephala0 aConformational transitions of cinnamoyl CoA reductase 1 from leu aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAR a30-350 v643 a

Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (L1-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 C and starts forming aggregates at 55 C. LI-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of Ll -CCRH1 at pH 2.0 was stable up to 90 C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCI), it got transformed into different structure which was stable in the vicinity of 2 M GdnHCI (as compared to drastic loss of native structure in 2 M GdnHC1) as seen in far UV-CD spectra. The structural transition of LI-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein. (C) 2013 Elsevier B.V. All rights reserved.

10aAggregation Cinnamoyl CoA reductase10aConformation10aMolten globule10aThermostability10aUnfolding1 aSonawane, Prashant, D.1 aKhan, Bashir, Mohammad1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/conformational-transitions-cinnamoyl-coa-reductase-1-leucaena-leucocephala-002010nas a2200229 4500008004100000022001400041245011000055210006900165260007300234300001200307490000700319520113100326653004101457653001801498653002601516100001701542700001601559700002501575700002401600700002701624856012901651 2014 eng d a0141-813000aLeucaena sp recombinant cinnamyl alcohol dehydrogenase: purification and physicochemical characterization0 aLeucaena sp recombinant cinnamyl alcohol dehydrogenase purificat aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcFEB a254-2600 v633 a

Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LICAD2) isolated from Leucaena leucocephala. LICAD2 is 80 kDa homodimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with K-cat/K-m of 11.6 x 10(6) (M-1 s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 C, with the deactivation rate constant (K-d*) and half-life (t(1/2)) of 0.002 and 5 h, respectively. LICAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (K-d*) and half-life (t(1/2)) of 7.5 x 10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn2+ per dimer, however, was inhibited in presence of externally supplemented Zn2+ ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents. (C) 2013 Elsevier B.V. All rights reserved.

10aCinnamyl alcohol dehydrogenase (CAD)10aMetalloenzyme10aSubstrate specificity1 aPatel, Parth1 aGupta, Neha1 aGaikwad, Sushama, M.1 aAgrawal, Dinesh, C.1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/leucaena-sp-recombinant-cinnamyl-alcohol-dehydrogenase-purification-and-physicochemical-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-002755nas a2200253 4500008004100000022001400041245012600055210006900181260007500250300001200325490000700337520179300344653002402137653002802161653001702189653001902206653002102225100002302246700003202269700001802301700002502319700002702344856013002371 2014 eng d a1053-050900aSteady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants0 aSteady state fluorescence studies of wild type recombinant cinna a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAY a665-6730 v243 a

Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K (a) = 3.72 x 10(5) M-1) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100 % of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f (a) = 1.0) was significantly higher than cesium ions (f (a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f (a) = 0.54), whereas quenching with iodide ion was decreased (f (a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions.

10aActive site mutants10aCinnamoyl CoA reductase10afluorescence10aLigand binding10aSolute quenching1 aSonawane, Prashant1 aVishwakarma, Rishi, Kishore1 aSingh, Somesh1 aGaikwad, Sushama, M.1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/steady-state-fluorescence-studies-wild-type-recombinant-cinnamoyl-coa-reductase-ll-ccrh1-002316nas 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

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.

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

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.

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

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.

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

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-002479nas a2200241 4500008004100000022001400041245010300055210006900158260005700227300000800284490000700292520161300299653002101912653001501933653002201948653003301970653002002003100001902023700001802042700002702060700002202087856012802109 2015 eng d a1610-294000aIn silico ligand binding studies of cyanogenic beta-glucosidase, dhurrinase-2 from Sorghum bicolor0 aIn silico ligand binding studies of cyanogenic betaglucosidase d a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcJUL a1840 v213 a

Dhurrinase, a cyanogenic beta-glucosidase from Sorghum bicolor is the key enzyme responsible for the hydrolysis of dhurrin to produce toxic hydrogen cyanide, as a part of plant defence mechanism. Dhurrinase 1 (SbDhr1) and dhurrinase 2 (SbDhr2), two isozymes have been isolated and characterized from S. bicolor. However, there is no information in the literature about the three dimensional (3D) structure of SbDhr2 and molecular interactions involved between the protein and ligand. In this study, the three dimensional structure of SbDhr2 was built based on homology modeling by using the X-ray crystallographic structure of its close homologue SbDhr1 as the template. The generated 3D model was energy minimized and the quality was validated by Ramachndran plot, various bioinformatic tools and their relevant parameters. Stability, folding-unfolding and flexibility of the modeled SbDhr2 was evaluated on the basis of RMSD, radius of gyration (R-g) and RMSF values respectively, obtained through molecular dynamic (MD) simulation. Further, molecular docking was performed with its natural substrate dhurrin, one substrate analogue, three un-natural substrates, and one inhibitor. Analysis of molecular interactions in the SbDhr2-ligand complexes revealed the key amino acid residues responsible to stabilize the ligands within the binding pocket through non-bonded interactions and some of them were found to be conserved (Glu239, Tyr381, Trp426, Glu454, Trp511). Reasonably broader substrate specificity of SbDhr2 was explained through the wider entrance passage observed in comparison to SbDhr1.

10abeta-Glucosidase10aDhurrinase10aMolecular docking10aMolecular dynamic simulation10aSorghum bicolor1 aMahajan, Chavi1 aPatel, Krunal1 aKhan, Bashir, Mohammad1 aRawal, Shuban, S. uhttps://library.ncl.res.in/content/silico-ligand-binding-studies-cyanogenic-beta-glucosidase-dhurrinase-2-sorghum-bicolor-102312nas a2200253 4500008004100000022001400041245010500055210006900160260007400229300001200303490000800315520137900323653003001702653001501732653002201747653002301769653001701792100001601809700001701825700003201842700003701874700002701911856012001938 2015 eng d a0167-685700aOverexpression of squalene synthase in withania somnifera leads to enhanced withanolide biosynthesis0 aOverexpression of squalene synthase in withania somnifera leads  aVAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDSbSPRINGERcAUG a409-4200 v1223 a

Genetic engineering of secondary metabolic pathways is an emerging area of research for production and improvement of natural products in plant biotechnology. Here, we describe a systematic approach to manipulate a key regulatory step of isoprenoid biosynthetic pathway in Withania somnifera to study its effect on withanolide production. We generated T-0 W. somnifera plants overexpressing squalene synthase (WsSQS) by Agrobacterium tumefaciens mediated transformation, which were analyzed by Gus biochemical assay and PCR of hygromycin phosphotransferase (hptII) and WsSQS. qRT-PCR analyses of various transformed tissues indicated 2-5 fold increase in WsSQS transcripts in both T-0 and T-1 generations. The tissue specific protein expression studies revealed 2-3 fold increase in WsSQS, which was further confirmed by enzyme activity. These observations were corroborated with the 1.5-2 fold increase in total withanolide content of the transformed tissues. However, in leaf tissue, the levels of Withaferin A and Withanolide A increased significantly up to 4-4.5 fold. These findings demonstrate genetic engineering of isoprenoid pathway in W. somnifera resulting in enhanced production of withanolides, and also provide insights into such metabolic pathways for their manipulation to improve the pharmacological content of different medicinally important plants.

10aAgrobacterium tumefaciens10aSolanaceae10aSqualene synthase10aWithania somnifera10aWithanolides1 aPatel, Neha1 aPatel, Parth1 aKendurkar, Shuchishweta, V.1 aThulasiram, Hirekodathakallu, V.1 aKhan, Bashir, Mohammad uhttps://library.ncl.res.in/content/overexpression-squalene-synthase-withania-somnifera-leads-enhanced-withanolide-002418nas a2200253 4500008004100000022001400041245010700055210006900162260007300231300001200304490000700316520147800323653001601801653002001817653002701837653004101864653001401905100002101919700001801940700002701958700002701985700002502012856012702037 2016 eng d a0141-813000aFunctional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera0 aFunctional and conformational transitions of mevalonate diphosph aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcFEB a160-1700 v833 a

Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40 degrees C and transient exposure of hydrophobic residues at 50 degrees C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher K-SV for IC as compared to that for CsCl. The native enzyme displayed two different lifetimes, vi (1.203 +/- 0.036 ns) and tau 2 (3.473 +/- 0.12 ns) indicating two populations of tryptophan. (C) 2015 Elsevier B.V. All rights reserved.

10aAggregation10aCD spectroscopy10aFluorescence quenching10aMevalonate diphosphate decarboxylase10aUnfolding1 aShakeel, Abbassi1 aPatel, Krunal1 aKhan, Bashir, Mohammad1 aBhosale, Siddharth, H.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/functional-and-conformational-transitions-mevalonate-diphosphate-decarboxylase-bacopa-0