We report the molecular cloning and characterization of a full-length cDNA probably encoding CNMT from Aristolochia fimbriata. The work presented here details the structural analysis of a translated nucleotide sequence from Arsitolochia fimbriata enzymes that could be involved in alkaloid biosynthesis. On the basis of computational analysis of the protein sequence it was predicted to be a SAM dependent S-coclaurine N-Methyltransferase, which transfers the activated methyl group of SAM to S-coclaurine. In this study three dimensional homology model has been analysed and the function of the translated sequence has been predicted.
In Glycine N-methyltransferase (GNMT), which catalyzes the S-adenosyl-L-methionine- (SAM-) dependent methylation of glycine producing sarcosine, SAM molecules bind to the SAH binding site . It has been previously found that the carboxylate and amino groups of the Met moiety of SAM in GNMT are involved in hydrogen bonding with Trp30, Arg40, Ala64, and Leu136. The OH of Tyr21 is positioned in such a way to have a charge-dipole interaction with the positively charged SD atom of SAM . In case of Aristolochia predicted CNMT, the carboxylate and amino groups of Met moiety of SAM are involved in hydrogen bonding with Glu-96, Ser-97, Gly-135 and Ile-201 (Fig. 3.39, 4.4 and 4.5). The OH of Tyr-79 (Fig. 4.2 and 4.3) is also positioned and oriented in such a way to make interactions with the positively charged SD atom of SAM which facilitates the transfer of methyl group. The substrate is surrounded by four phenyl rings and a guanidinium ring of His in such a way that it makes a pocket that just suits the S-Coclaurine and this also agrees with the proposal that a shorter side chain is necessary to accommodate the larger substrates of the N-methyltransferases . One of the four Phe surrounding the S-Coclaurine (Phe-256) is conserved in all CNMTs as shown by the multiple alignment (Fig. 3.48). In GNMTs Tyr-21 has been implicated in its methyl transfer mechanism  however in CNMTs Tyr-79 (As numbered in Aristolochia CNMT) is conserved (Fig. 3.48) and its hydroxyl group is in close proximity to the positively charged SD atom of SAM as has been previously stated about motif B methyltransferases . From this we infer that the Tyr-79 (numbering is different for different CNMTs) might be involved in transfer of methyl group in CNMTs of plants (Fig. 4.2 and 4.3).
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4.2: Conserved residues of CNMTs.
Multiple alignment of all the CNMTs selected (Fig. 3.48) shows that residues E-16, P-21, R-30, L-33, R-36, Y-79, P-82, G-91, K-95, S-97, L-108, A-111, E-112, L-116,118, Y-119, 121-ERA-123, G-128, L-132, 135-GCG-137, G-139, L-143, A-146, S-160, Q-163, K-164, I-167, N-178, 196-DR-197, 205-EHMKNY-210, 213-LL-214, W-220, 228-LF-229, H-235, Y-240, E-243, 249-DW-250, F-256, G-259, S-265, 270-LYFQ-273, W-282, G-286, H-288, E-294, W-296, D-301, A-321, 328-WR-329, 339-F, Y-341, G-344, 346-EW-347, H-351, and 353-LF-354, are conserved in all selected CNMTs. The last three residues of Lys are conserved in all except Chlamydomonas. In Oryza putative CNMT, only one of the three Lys residues is different. If we compare these conserved residues with the SAM binding residues, we find that most of the SAM binding residues are among these conserved residues. Only one residue i.e. F-256 among S-Coclaurine binding residues is found among the conserved residues. Other substrate and SAM binding residues are also conserved, although the residues are different in some of the CNMTs, but those different residues are characterically similar to one another, therefore we can say that they are also conserved.
As no CNMT crystalline structure has been determined yet therefore we selected other methyltransferases for comparison. Xanthosine (XR) and S-Coclaurine are very similar in their 3D structures, so we compare the substrate binding residues of xanthosine methyltransferase (XMT), dimethylxanthine methyltransferase (DXMT) and salicyclic acid O-methyltransferase (SAMT). In XMT Ser-316 (Val in DXMT and Cys in SAMT) makes a hydrogen bond with the O5` OH group of the Rib moiety of XR, but Val cannot make this bond. Tyr-356 of XMT (Tyr-368 in DXMT) interacts with the O2 carboxyl group of XR through hydrogen bonding. Similarly Tyr-321 in XMT interacts with the O2 carboxyl group of XR through a hydrogen bond. In DXMT, the structurally conserved Tyr-333 is too far from any potential hydrogen-bonding partners and can not directly contribute to the binding of substrate . In SAMT this position is occupied by Ala-312 and is necessary to have the larger Trp-226, which is involved in forming a part of the salicylate-binding site of SAMT . In Aristolochia CNMT the substrate is surrounded by four Phe, one Gly one Glu and one Asp groups as described above. All the surrounding residues are arranged in such ways that form a pocket for coclaurine. The four Phe residues and the Gly cannot make hydrogen bonds, Asp is oriented in such a way that is unable to make a hydrogen bond. However the ND1 of His232 forms a hydrogen bond with O2 of S-Coclaurine. Similarly OE1 of Glu96 forms a Hydrogen bond with N4 (the nitrogen to be methylated) of the coclaurine. This last residue seems to play important role in the transfer of methyl group.
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4.3: Overall structure
In order to elucidate the mechanism of substrate binding, homology modelling of CNMT complexed with SAM was performed and Coclaurine was docked into the active site.
The model built has already been explained in the result section. The structure can be divided into two domains: the N-terminal catalytic core domain and the C-terminal domain. The catalytic domain comprises a central strand of twisted ï¿½-sheets flanked by alpha helices on both the sides. The catalytic core domain contains the binding sites for SAM. The C-terminal domain contains alpha helices making a roof over the substrate and a few beta sheets forming the base for the substrate.
4.3.1: SAM/SAH binding site
S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) are metabolites that are involved in the conversion of methionine to homocysteine in the proximal end of the methionine cycle. S-Adenosylmethionine is synthesized from methionine and ATP in a reaction catalysed by methionine adenosyltransferase. The methyl group is provided by SAM which is also involved in several dozens of transmethylation reactions of crucial biological importance .
The glycine-rich sequence containing Gly-72 and Gly-74 ((E/ D)XGXGXG), often referred to as motif I, is highly conserved in many SAM-dependent methyltransferases and is also present in TNMTs, CNMTs, and MtPcaA [188, 311, 367]. MtPcaA exhibits a structural fold which is most similar to the small molecule subclass of methyltransferases . The conservation of critical residues involved in SAM-binding suggests that TNMT and CNMTs also maintain this core small molecule methyltransferase fold. A methyltransferase fold model also predicts that SAM-binding motifs happen in the N-terminal domains of TNMT and CNMTs. As such, amino acid residues that confer substrate specificity are most likely situated in the C-terminal regions of these proteins . In the absence of crystal structures for CNMTs and TNMT, residues that confer substrate and reaction specificity with respect to nitrogen methylation might be identified among C-terminal residues that are conserved in CNMTs but are unique to TNMT.
As it is clear from the homology model of CNMT that SAM/SAH has an extended conformation assuming the binding site to be identical  for both SAM and SAH, therefore we discuss below the structure of the CNMT-SAM complex as a representative of both complexes. SAM/SAH binds to the pocket that is long and wide enough to hold the ligand. This pocket is located near the substrate binding pocket where the donor-methyl group protrudes outward from the substrate pocket towards the ligand binding pocket (Fig. 4.1). This type of situation is almost similar to the binding of SAM in Streptococcus pneumoniae Sp1610 (a putative tRNA methyltransferase) . The bound SAM is located on the inner surface of a large cavity (Fig. 4.1). SAM binds to Glu-96, Ser-97, Gly-135, Gly-137, Thr-158, Asn-159, Gln-163, Ile-186 and Ile-201, as well as interacting with Tyr-79, Leu-134, Val-157, Asp-185, Leu-203, His-206 and Phe-256 through weak Van der Wallï¿½s interaction (Fig. 3.39). However In case of Sp1610 the SAM is stabilized by Glu46 and Arg5 by forming hydrogen and ionic contacts with SAM and is stabilized further by several residues in the binding pocket. The adenine moiety of SAM makes hydrophobic contacts with Val22, Val47, Asn74, Leu97, Met93, and Ile101 while the N6 atom of adenine and Asn74 forms a hydrogen bond and the hydroxyl groups in the ribose ring form hydrogen bonds with Glu46. The carboxyl groups of SAM in Sp1610 are recognized by Arg5 with the chargeenhanced hydrogen bond and the amino groups of SAM are in contact with the backbone carbonyl groups of Gly23 and Ala91 through hydrogen bondings .
The Aristolochia CNMT shows a total of 10 hydrogen bonds between CNMT and SAM (Fig. 3.39), which seems to fix exactly the position and orientation of SAM. Notably, the O atom of Tyr-79 and the sulfur atom of SAM are positioned in close vicinity (Fig. 4.2 and 4.3). Most of the MTases have been observed to posses the interactions between the sulfur atom of SAM/SAH and an oxygen atom of an amino acid of the enzyme  for example in rRNA N6-MTase, the interaction between the sulfur atom of SAM and an oxygen atom of a main chain Asn residue , and in histone-lysine N-MTase, the interaction between the sulfur atom of SAH and the Od atom of an Asn residue . These interactions are required to be studied further for the elucidation of their precise role.
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4.3.2: Substrate binding pocket
There is a large cavity between the two domains of CNMT, which is large enough to hold the substrate, Coclaurine (Fig. 3.41 and 3.42). Moreover, the SAM-binding site is located next to this cavity and there is an opening in between these two cavities through which the methyl group of SAM is projecting towards this cavity, suggesting strongly that Coclaurine binds to this cavity. The substrate is oriented in such a way that the Nitrogen atom of Coclaurine to be methylated comes just in front of the opening that connects the two cavities, which is just suitable for the transfer of the methyl group to the Coclaurine (Fig. 3.44 and 4.1). Coclaurine binds to Glu-96 and His-232 through hydrogen bonds, as well as interacting with Gly-202, Asp-231, Phe-251, Phe-256, Phe-325 and Phe-349 through weak Van der Wallï¿½s interactions (Fig. 3.45). There are a total of 2 hydrogen bonds between CNMT and Coclaurine, which fix exactly the position and orientation of Coclaurine. Notably, the OE1 atom of Glu-96 and the N4 atom of Coclaurine are positioned in close vicinity forming a hydrogen bond (Fig. 4.4 and 4.5) that might facilitate the transfer of methyl to Coclaurine.
A large class of SAM-dependent methyltransferases has been found to share a conserved catalytic domain structure due to the interaction of the enzymes with a common cofactor, S-adenosylmethionine . It has been shown that SAM-dependent methyltransferases show a similar folding pattern with a central parallel b-sheet surrounded by alpha-helces [347, 421-428]. The common three-dimensional structure of these enzymes is reflected in sequence motifs that are conserved among a large number of SAM-dependent methyltransferases [429-434].
In general, MTases posses three conserved binding motifs: motif 1 containing the GXGXGG sequences is involved in binding to the amino acid portion; motif 2 having an acidic and a hydrophobic residue, the acidic reisdue is responsible for making bonds to the ribose moiety and the hydrophobic residue is involved in forming the hydrophobic pocket to stabilize the adenine ring while motif 3 make contacts to the adenine ring via ionic or hydrogen bonds with D/E/N/Q residues .
The conserved regions; motifs I, post-I, II, and III are always found in the same order on the polypeptide chain and are separated by comparable intervals . The three-dimensional structures of AdoMet-dependent methyltransferases have shown that motif I and post-I interact directly with the SAM residue, while motifs II and III make contacts with each other and with a portion of motif I to form the main central portion of the b-sheet.
The SAM-dependant methylation reaction involves the direct transfer of the methyl group from SAM to the substrates, and thus the cavities for both the ligand and substrate should be positioned close to each other and oriented in such a way that they can transfer the methyl group easily . As we know that in CNMT methyl transfer reaction is dependent on SAM we propose that CNMT catalyzes the methyltransfer reaction by ï¿½proximity and orientation effectsï¿½ just like GNMT does . On the basis of the arrangement of the conserved residues in the CNMT cavity, the location and orientation of the bound SAM, and the proposed binding mode of Coclaurine, we propose a reaction mechanism for the CNMT as follows. In general, MTases in general are thought to to be involved in the direct transfer of the methyl group to the substrate with inversion of symmetry by an SN2-like mechanism . Applying the SN2-like reaction concept in this case, the reaction catalyzed by CNMT has been proposed to occur as described in the following lines. The OH of Tyr79 is positioned to have a charge-dipole interaction with the positively charged SD of SAM (Fig. 4.2 and 4.3). Similarly Glu96 is positioned in such a way that it can take part in the removal of the hydrogen atom from N4 of coclaurine (Fig. 4.4 and 4.5).
A possible mechanism for methylation of Coclaurine seems to be that the OH of Tyr79 induces positive charge in SD atom of SAM. First, the hydroxyl group of Tyr-79 is possible to be deprotonated. His-206 may act as a base to accept the proton from Tyr-79 . Second, the resulting oxyanion of Tyr-79 may interact with SD atom of SAM to form to make Van der Walls interaction. This helps in loosning the bond between SD atom of SAM and the methyl group. Interactions between the sulfur atom of SAM/SAH and an oxygen atom of an enzyme have been observed in other MTases as well [346, 347, 419], however in some cases the situation is different for example BchU where the Nd atom of His150 and the sulfur atom of SAH are positioned in close vicinity (3.1 ï¿½) which was proposed to be the first example of the case . In our case His-206 is in close proximity to the SD atom of SAM, but the distance is 5.28ï¿½ which is greater than that of BchU. The positive charge induced on SD atom of SAM also induces positive charge on the carbon atom of methyl group attached to the SD atom of SAM. The negatively charged unshared pair of electrons on N4 atom of coclaurine is attracted by the positively charged methyl group of SAM. The carboxyl oxygen of the Glu-96 seems to deprotonate the N4 atom of Coclaurine. In the mean time the bond between methyl group and SD atom of SAM is broken and a new bond is formed between the methyl group and N4 atom of coclaurine and the hydrogen seems to be taken by Glu96 and in this way methylation of coclaurine happens by the SAM. The substrates for the O-methyltransferases are expected to be fully or predominantly deprotonated at cellular pH values and should only require their correct positioning for methylation to occur . Tyr-79 noted earlier could form a charge dipole interaction with the positively charged SD atom of SAM, facilitating the methyl transfer reaction in this family of methyltransferases, as observed in Gly N methyltransferases . The deprotonation of N4 atom with the concomitant cleavage of the Cï¿½S bond in SAM and a new bond formation between the methyl group and the N4 atom of Coclaurine seems to happen simultaneously and hence follows the SN2 mechanism. The N-methyltransferases may also require some additional help for methylation because nitrogen is not as electronegative as oxygen .
4.5: Phylogenetic Analysis:
NJ consensus tree based on 1000 bootstrap, rooted with MACPS was created to know about the relationship of Aristolochia with other closely related species and to find out about the function of the gene isolated from Aristolochia (Fig. 3.46).
Although TNMT and CNMTs share limited sequence similarity with cyclopropane synthases, several residues known to be involved in SAM binding in MtPcaA (bacterial SAM-dependent cyclopropane fatty acid synthases) are strictly conserved  as explained above. The phylogenetic tree revealed that the Aristolochia sequence was similar to other CNMTs obtained from other plants as they appeared in the same cluster, but the cluster does not look monophyletic due to the clustering of TNMTs with the sequence which can also mean that TNMTs might be very closely related to CNMTs and they might have evolved from a single ancestor. Phylogenetic analysis also shows a monophyletic origin of the two N-methyltransferases i.e. CNMT and TNMT . The metabolic role suggests that TNMT has appeared by the duplication of a gene after the more ancient recruitment of CNMT, which is supported by the widespread occurrence of TNMT activity in the Papaveraceae but not in other members of plant families that are involved in the accumulation of benzylisoquinoline alkaloids . The glycine-rich sequence containing Gly-72 and Gly-74 ((E/D)XGXGXG), often referred to as motif I is highly conserved in many SAM-dependent methyltransferases and is also present in TNMT, CNMTs, and MtPcaA [188, 311, 367]. Structure and sequence conservation shows that MtPcaA exhibits a structural fold most similar to the small molecule subclass of methyltransferases .
The Aristolochia sequence and CNMTs of Papaver, Coptis and Thalictrum are supported with a high bootstrap value of 90. This high support reveals that the Aristolochia sequence might be the CNMT sequence and may be involved in transferring of a methyl group from SAM to S-Coclaurine.
A MP tree rooted with Chlamydomonas CNMT was generated from the multiple aligned CNMT sequences of Aristolochia and other CNMTs from plants whose CNMT sequences are already known to find out about their relationship. The tree reveals a closer relationship of Aristolochia with Coptis and Thalictrum as compared to Arabidopsis and Oryza as supported by the high bootstrap value of Aristolochia CNMT with Coptis and Thalictrum CNMTs.
Arabidobsis has a closer relation to Orysa as the tree shows that they have a very strong support of 91. A very high bootstrap support of 100 for Coptis and Thalictrum shows that Coptis and Thalictrum might have evolved recently from one another having a recent common ancestor.