Molecular cloning and characterization of genistein 4′-O-glucoside specific glycosyltransferase from Bacopa monniera

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...

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Bibliographic Details
Published in:Molecular biology reports 2014-07, Vol.41 (7), p.4675-4688
Main Authors: Ruby, Santosh Kumar, R. J., Vishwakarma, Rishi K., Singh, Somesh, Khan, Bashir M.
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Animal Anatomy
Animal Biochemistry
Bacopa - classification
Bacopa - drug effects
Bacopa - enzymology
Bacopa - genetics
Base Sequence
Benzopyrans - metabolism
Biomedical and Life Sciences
Cloning
Cloning, Molecular
Enzymes
Escherichia coli - genetics
Escherichia coli - metabolism
Gene Expression
Glycosyltransferases - chemistry
Glycosyltransferases - genetics
Glycosyltransferases - metabolism
Histology
India
Life Sciences
Lycium - chemistry
Lycium - enzymology
Molecular biology
Molecular Sequence Data
Morphology
Phylogeny
Plant biology
Plant Leaves - drug effects
Plant Leaves - enzymology
Plant Leaves - genetics
Plant Proteins - chemistry
Plant Proteins - genetics
Plant Proteins - metabolism
Plant Roots - drug effects
Plant Roots - enzymology
Plant Roots - genetics
Plant Stems - drug effects
Plant Stems - enzymology
Plant Stems - genetics
Plants, Medicinal
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Salicylic Acid - pharmacology
Sequence Alignment
Substrate Specificity
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Summary: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.
ISSN:0301-4851
1573-4978
DOI:10.1007/s11033-014-3338-8