Title of Thesis

Sequencing And Analysis of A Cdna Encoding A Putative Coclaurine N-methyltransferase (cnmt) From Aristolochia Fimbriata, A Basal Angiosperm

Author(s)

Roshan Ali

Abstract
Alkaloids are produced in plants through various pathways involving several enzymes that lead to diverse alkaloids. One of the most important alkaloid biosynthetic enzymes is coclaurine N-methyltransferase (CNMT) which is an S-adenosyl-L-methionine-dependent methyltransferase (SAM-MTase). SAM-MTases utilize S-adenosyl-L-methionine (SAM) as a cofactor to methylate other molecules. CNMT catalyzes the methylation of coclaurine. Crystal structures of more than hundred SAM-MTases have been investigated. Several O-methyltransferases have been characterized at the molecular as well as structural levels, but there have been very few molecular studies of N-methyltransferases especially about CNMTs.
In this study, the amino acids sequence of Aristolochia fimbriata putative CNMT has been determined by isolating and translating the full-length cDNA. In order to investigate the mechanism of methylation by this putative CNMT, three-dimensional homology model has been built and the ligand (SAM) as well as the substrate (S-Coclaurine) has been docked into its active site. Phylogenetic analyses were performed using the MEGA 4.0 software. The phylogenetic relationship of A. fimbriata putative CNMT with their homologs has also been analyzed. In order to identify the putative CNMT gene and determine its function, online similarity searches were performed by BLAST program using the cDNA sequence as well as the putative protein that could be encoded by the gene. All the methods, applied, predicted that the gene identified might be involved in the production of CNMT.
The predicted homology model consists of two domains: the N-terminal catalytic core domain and the C-terminal domain. The catalytic core domain has a central sheet of β-strands surrounded by α helices. The catalytic core domain contains binding site for SAM. The C-terminal domain consists of alpha helices and a few beta sheets creating a pocket for the substrate in between them. The SAM-binding pocket is located next to substrate binding pocket and there is an opening in between these two cavities through which the methyl group of SAM projects towards the substrate. The most important residues involved in the methyl transfer reaction seem to be Tyr-79 and Glu-96.

1

1.1 S-adenosyl-L-Methionine-Dependent Methyltransferases
1.2 S-Adenosyl-L-Methionine (SAM or AdoMet)
1.3 Genes of Bezylisoquinoline Alkaloid Pathway
1.4 Alkaloids
1.5 Aristolochia
1.6 Aim of the Present Study

2.1 Tissue Source
2.2 RNA Extraction
2.3 Quality and Purity of the Total RNA
2.4 Messenger RNA (mRNA) Isolation
2.5 Unigene Selection from A. fimbriata EST Database
2.6 Analysis and Amplification of Gene Expression by Reverse Transcriptase RT-PCR
2.7 Gene Identification and Prediction of the Function of its Putative Protein
2.8 Homology Model Building
2.9 Active Site Identification
2.10 Docking
2.11 Analysis of Ligand and Substrate Binding Interactions with the Active Site Residues
2.12 Phylogenetic Analysis

3.1 Qualitative Assessment of Extracted Total RNA
3.2 PCR and Gel Extraction
3.3 Nucleotide Sequence Chromatogram Analysis
3.4 Tribe Identification
3.5 Identification of the Gene and Prediction of the Putative Function of its Protein
3.6 Homology Model Building
3.7 3D Structure of the Model
3.8 Comparison with Theoretical Models of Other CNMTs
3.9 Binding Site
3.10 Putative Reaction Mechanism
3.11 Phylogenetic Analysis