Molecular and biochemical studies on components of transcription regulation, tropane alkaloid biosynthesis and signal transduction in the medicinal plant Datura metel L.

Abstract

Tropane alkaloids (TA) are among the oldest medicines known to humankind as they possess hallucinogenic and poisonous properties. Datura metel, an herbaceous plant, belongs to the family Solanaceae whose members are well known to produce tropane alkaloids. The isolation of the key biosynthetic enzymes, transcription factors, and protein kinases and their corresponding genes, is important in understanding TA biosynthesis and in developing metabolic engineering approaches to enhance the amount of tropane alkaloids in plants. Putrescine-N- methyltransferase (PMT; EC 2.1.1.53) is the first enzyme in the tropane alkaloid and nicotine biosynthetic pathway and it catalyzes the S-adenosylmethionine (SAM) dependent N-methylation of putrescine to methyl-putrescine. In the present study, Datura metel putrescine methyltransferase (DmPMT) protein has been biochemically and biophysically characterized. The initial rate kinetic study for DmPMT showed the Vmax 24.47 nKat/mg, Km for Putrescine 255.5 M and Km for SAM were 128.2M. The ordered Bi Bi kinetic mechanism best describes the N-methyl-transferase reaction catalyzed by DmPMT. Upon structural analysis of this N-terminus β-sheet and putrescine binding pocket, we could predict that the interaction of Trp-64 of N-term to the amino group of Putrescine through Cation-π interaction. Most probably, this interaction is crucial for keeping Putrescine in better orientation towards SAM. Here we report the isolation of transcription factor DORA and DmMYB from Datura metel. q-RTPCR showed that expression of the DORA gene was strongly induced by treatment with fungal extract and moderately induced by methyl jasmonate plus wounding treatments. The Gel mobility shift assay showed that the recombinant DORA protein could bind specifically to GCC box, a cis-acting element present in the promoter regions of several target genes regulated by AP2 domain-containing transcription factor. DmMYB also exhibited sequence-specific DNA-binding. The binding constant for DmMYB-DNA interaction (Kd) was calculated to be 8.1*10-9 M, and the stoichiometry of the interaction was 1:1. 3D homology model of the DmMYB-DNA complex was constructed, confirming the role of TRP-17 and TRP-89 in complex formation. Further, our model highlighted a possible functional role of DmMYB-Mg2+ binding. In the present study, we have isolated a full-length CDPK cDNA, DmCDPK1 (GenBank accession number ABY28389), from Datura metel by RT-PCR and rapid amplification of cDNA ends (RACE) methods. The recombinant DmCDPK1 with His-tag was heterologous expressed in E. coli and purified using Ni-NTA affinity chromatography. Biochemical analyses showed that both activities of autophosphorylation and phosphorylation of histone III-S substrate by DmCDPK1 protein are dependent on calcium. The kinase activity of the recombinant enzyme was calmodulin independent and sensitive to CaM antagonists, W7, and calmidazolium.