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
VSAM. 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.
