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• W1991569676 abstract "S-Adenosyl-l-methionine:coclaurineN-methyltransferase (CNMT) converts coclaurine toN-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5′- and 3′-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-l-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that CoptisCNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution ofN-methyltransferases in secondary metabolism is discussed based on sequence similarity. S-Adenosyl-l-methionine:coclaurineN-methyltransferase (CNMT) converts coclaurine toN-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5′- and 3′-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-l-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that CoptisCNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution ofN-methyltransferases in secondary metabolism is discussed based on sequence similarity. S-adenosyl-l-methionine coclaurineN-methyltransferase N-methyltransferase O-methyltransferase liquid chromatography-mass spectrometry 5′ rapid amplification of cDNA ends S-Adenosyl-l-methionine (AdoMet)1:coclaurineN-methyltransferase (CNMT) (1Choi K.B. Morishige T. Sato F. Phytochemistry. 2001; 56: 649-655Crossref PubMed Scopus (34) Google Scholar, 2Frenzel T. Zenk M.H. Phytochemistry. 1990; 29: 3491-3497Crossref Scopus (44) Google Scholar, 3Wat C.K. Steffens P. Zenk M.H. Z. Naturforsch. 1986; 41c: 126-134Crossref Scopus (27) Google Scholar) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. This is a unique N-methyltransferase in the biosynthesis of benzylisoquinoline alkaloids (Fig.1). This enzyme is thought to be important because N-methylation of coclaurine strongly enhances the 4′-O-methylation activity of 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase and enables the sequential metabolic conversion of substrates (4Morishige T. Tsujita T. Yamada Y. Sato F. J. Biol. Chem. 2000; 275: 23398-23405Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 5Stadler R. Zenk M.H. J. Biol. Chem. 1993; 268: 823-831Abstract Full Text PDF PubMed Google Scholar). Furthermore, the enzymatic activity at this important step is rather low relative to the entire biosynthetic pathway (1Choi K.B. Morishige T. Sato F. Phytochemistry. 2001; 56: 649-655Crossref PubMed Scopus (34) Google Scholar, 6Sato F. Takeshita N. Fitchen J.H. Fujiwara H. Yamada Y. Phytochemistry. 1993; 32: 659-664Crossref Scopus (35) Google Scholar, 7Sato F. Tsujita T. Katagiri Y. Yoshida S. Yamada Y. Eur. J. Biochem. 1994; 225: 125-131Crossref PubMed Scopus (65) Google Scholar, 8Yamada Y. Okada N. Phytochemistry. 1985; 24: 63-65Crossref Scopus (33) Google Scholar). Thus, we purified CNMT and characterized its properties. Previous studies have clearly indicated that CNMT is non-stereospecific and has broad substrate specificity;Coptis enzyme methylated even simple dihydroxyisoquinoline alkaloids.Whereas several O-methyltransferases have been characterized at the molecular level (4Morishige T. Tsujita T. Yamada Y. Sato F. J. Biol. Chem. 2000; 275: 23398-23405Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 9Takeshita N. Fujiwara H. Mimura H. Fitchen J.H. Yamada Y. Sato F. Plant Cell Physiol. 1995; 36: 29-36PubMed Google Scholar, 10Ibrahim R.K. Trends Plant Sci. 1997; 2: 249-250Abstract Full Text PDF Google Scholar, 11Ibrahim R.K. Bruneau A. Bantignies B. Plant Mol. Biol. 1998; 36: 1-10Crossref PubMed Scopus (232) Google Scholar, 12Frick S. Kutchan T.M. Plant J. 1999; 17: 329-339Crossref PubMed Scopus (138) Google Scholar), there have been very few molecular studies of N-methyltransferases in secondary metabolite biosynthesis in plants (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar, 14Kato M. Mizuno K. Crozier A. Fujimura T. Ashihara H. Nature. 2000; 406: 956-957Crossref PubMed Scopus (156) Google Scholar). The differences in the primary structures, including theS-adenosyl-l-methionine binding site of NMTs and OMTs reported to date, suggest that molecular isolation of CNMT based on the structural similarity of methyltransferases would be very difficult. Thus, we adapted the conventional strategy to isolate cDNA based on the amino acid sequence of purified enzyme. Whereas our purified CNMT fraction still contained two protein bands of about 45 kDa, careful inspection of the chromatographic behavior of the proteins and enzyme activity suggested that the slow moving 45-kDa polypeptide would encode CNMT (1Choi K.B. Morishige T. Sato F. Phytochemistry. 2001; 56: 649-655Crossref PubMed Scopus (34) Google Scholar). Based on this observation, we determined the N-terminal amino acids, isolated the corresponding cDNA fragment, and finally the full-length cDNA. The nucleotide sequence of cDNA suggested that the deduced amino acid sequence showed some similarity to known NMTs such as phosphoethanolamine NMT. Further characterization of recombinant protein heterologously expressed in Escherichia coli confirmed that this isolated cDNA encoded CNMT. Recombinant CNMT was purified to homogeneity, and its enzymological properties were characterized. The evolution of CNMT involved in secondary metabolism is discussed based on the sequence similarity of this novel NMT.DISCUSSIONBased on the N-terminal amino acid sequence of purified CNMT and information on codon usage in Coptis genes, a full-length cDNA encoding CNMT was successfully cloned and its enzymatic activity was confirmed in a heterologous expression system. Recombinant CNMT purified from transgenic E. coli showed a significantly higher activity than that of CNMT purified from culturedCoptis cells. CNMT deduced from the cDNA sequence was 358 amino acids long with a calculated molecular mass of 41 kDa, which was slightly lower than the apparent molecular mass (45 kDa) of purified CNMT determined by SDS-PAGE. SDS-PAGE analysis of purified recombinant CNMT revealed a similar mobility for CNMT purified fromCoptis cells, indicating that this discrepancy between calculated molecular mass and apparent molecular mass was not due to post-translational modification but rather to experimental variation probably associated with the surface charge or conformation of CNMT.Because this CNMT sequence was the first reported NMT sequence in isoquinoline alkaloid biosynthesis and the third reported NMT in alkaloid biosynthesis in plants (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar, 14Kato M. Mizuno K. Crozier A. Fujimura T. Ashihara H. Nature. 2000; 406: 956-957Crossref PubMed Scopus (156) Google Scholar), the sequences of these plant alkaloid NMTs and other methyltransferases were compared to obtain more information about the diversity of methyltransferases. Whereas several plant O-methyltransferases have relatively high sequence homology ranging from 32 to 71%, the diversity ofN-methyltransferases is quite high (Fig. 3); the sequence identity among NMTs is about 5–15%. This means that each NMT may have evolved from an independent origin. CNMT showed relatively high homology to a hypothetical Arabidopsis protein (T05192) and cyclopropane-fatty acyl phospholipid synthase ofMesorhizobium (21Kaneko T. Nakamura Y. Sato S. Asamizu E. Kato T. Sasamoto S. Watanabe A. Idesawa K. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kiyokawa C. Kohara M. Matsumoto M. Matsuno A. Mochizuki Y. Nakayama S. Nakazaki N. Shimpo S. Sugimoto M. Takeuchi C. Yamada M. Tabata S. DNA Res. 2000; 7: 331-338Crossref PubMed Scopus (660) Google Scholar), whereas the functional similarity is not clear.The recent determination of the three-dimensional structures of plantO-methyltransferases suggested the structural conservation of an AdoMet binding site among methyltransferases (22Zubieta C. He X.Z. Dixon R.A. Noel J.P. Nat. Struct. Biol. 2001; 8: 271-279Crossref PubMed Scopus (271) Google Scholar). As Chandrashekhar and Vincent (23Chandrashekhar P.J. Vincent L.C. Plant Mol. Biol. 1998; 37: 663-674Crossref PubMed Scopus (234) Google Scholar) have deduced from 56 plant enzyme sequences, motif A (V/I/L)(V/L)(D/K)(V/I)GGXX(G/A) is present in all plant O-methyltransferases with 0–2 mismatches. Motifs B and C, (V/I/F)(A/P/E)X(A/P/G)DAXXXK(W/Y/F) and (A/P/G/S)(L/I/V)(A/P/G/S)XX(A/P/G/S)(K/R)(V/I)(E/I)(L/I/V) respectively, are conserved 98% with 0–3 mismatches (X is any amino acid). On the other hand, the AdoMet recognition domain was not as highly conserved in NMTs, even though motif A was found among almost all OMTs. In CNMT, only motif A is present with two mismatches (Fig. 6). The lower conservation of the AdoMet binding domain in NMT might be due to structural differences between NMTs and OMTs. Whereas many OMTs have an AdoMet binding site in their C-terminal half, motif A in CNMT and other NMTs is located at the N-terminal end (24Quaife C.J. Hoyle G.W. Froelick G.J. Findley S.D. Baetge E.E. Behringer R.R. Hammang J.P. Brinster R.L. Palmiter R.D. Transgenic Res. 1994; 3: 388-400Crossref PubMed Scopus (9) Google Scholar, 25Ying Z. Mulligan R.M. Janney N. Houtz R.L. J. Biol. Chem. 1999; 274: 36750-36756Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 26Scoilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (58) Google Scholar). Interestingly, a putativeArabidopsis protein (T05192) also has this motif A, and cyclopropane-fatty acyl phospholipid synthase also shows a similar identity in this region. Some enzymes in secondary metabolism such as putrescine N-methyltransferase and tropinone reductase have been postulated to have evolved from enzymes in primary metabolism such as spermine synthase or the short-chain dehydrogenase gene family (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar,27Nakajima K. Hashimoto T. Yamada Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9591-9595Crossref PubMed Scopus (118) Google Scholar). Further characterization of the enzyme activity ofArabidopsisT05192 and site-directed mutagenesis of CNMT should provide clues for understanding the evolution of CNMT.Figure 6Amino acid sequence alignment of the motif A domains of the N- andO-methyltransferases in C. japonica, a hypothetical protein in A. thaliana, and cyclopropane-fatty acyl phospholipid synthase in M. loti. Motif A is a conserved sequence motif in plantS-adenosyl-l-methionine-dependent methyltransferases. Amino acids conserved in all of the sequences areboxed, and similar ones are shaded. Cyclopropane, cyclopropane-fatty acyl phospholipid synthase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; 6OMT, norcoclaurine 6-O-methyltransferase; SMT, scoulerine 9-O-methyl-transferase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) S-Adenosyl-l-methionine (AdoMet)1:coclaurineN-methyltransferase (CNMT) (1Choi K.B. Morishige T. Sato F. Phytochemistry. 2001; 56: 649-655Crossref PubMed Scopus (34) Google Scholar, 2Frenzel T. Zenk M.H. Phytochemistry. 1990; 29: 3491-3497Crossref Scopus (44) Google Scholar, 3Wat C.K. Steffens P. Zenk M.H. Z. Naturforsch. 1986; 41c: 126-134Crossref Scopus (27) Google Scholar) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. This is a unique N-methyltransferase in the biosynthesis of benzylisoquinoline alkaloids (Fig.1). This enzyme is thought to be important because N-methylation of coclaurine strongly enhances the 4′-O-methylation activity of 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase and enables the sequential metabolic conversion of substrates (4Morishige T. Tsujita T. Yamada Y. Sato F. J. Biol. Chem. 2000; 275: 23398-23405Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 5Stadler R. Zenk M.H. J. Biol. Chem. 1993; 268: 823-831Abstract Full Text PDF PubMed Google Scholar). Furthermore, the enzymatic activity at this important step is rather low relative to the entire biosynthetic pathway (1Choi K.B. Morishige T. Sato F. Phytochemistry. 2001; 56: 649-655Crossref PubMed Scopus (34) Google Scholar, 6Sato F. Takeshita N. Fitchen J.H. Fujiwara H. Yamada Y. Phytochemistry. 1993; 32: 659-664Crossref Scopus (35) Google Scholar, 7Sato F. Tsujita T. Katagiri Y. Yoshida S. Yamada Y. Eur. J. Biochem. 1994; 225: 125-131Crossref PubMed Scopus (65) Google Scholar, 8Yamada Y. Okada N. Phytochemistry. 1985; 24: 63-65Crossref Scopus (33) Google Scholar). Thus, we purified CNMT and characterized its properties. Previous studies have clearly indicated that CNMT is non-stereospecific and has broad substrate specificity;Coptis enzyme methylated even simple dihydroxyisoquinoline alkaloids. Whereas several O-methyltransferases have been characterized at the molecular level (4Morishige T. Tsujita T. Yamada Y. Sato F. J. Biol. Chem. 2000; 275: 23398-23405Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 9Takeshita N. Fujiwara H. Mimura H. Fitchen J.H. Yamada Y. Sato F. Plant Cell Physiol. 1995; 36: 29-36PubMed Google Scholar, 10Ibrahim R.K. Trends Plant Sci. 1997; 2: 249-250Abstract Full Text PDF Google Scholar, 11Ibrahim R.K. Bruneau A. Bantignies B. Plant Mol. Biol. 1998; 36: 1-10Crossref PubMed Scopus (232) Google Scholar, 12Frick S. Kutchan T.M. Plant J. 1999; 17: 329-339Crossref PubMed Scopus (138) Google Scholar), there have been very few molecular studies of N-methyltransferases in secondary metabolite biosynthesis in plants (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar, 14Kato M. Mizuno K. Crozier A. Fujimura T. Ashihara H. Nature. 2000; 406: 956-957Crossref PubMed Scopus (156) Google Scholar). The differences in the primary structures, including theS-adenosyl-l-methionine binding site of NMTs and OMTs reported to date, suggest that molecular isolation of CNMT based on the structural similarity of methyltransferases would be very difficult. Thus, we adapted the conventional strategy to isolate cDNA based on the amino acid sequence of purified enzyme. Whereas our purified CNMT fraction still contained two protein bands of about 45 kDa, careful inspection of the chromatographic behavior of the proteins and enzyme activity suggested that the slow moving 45-kDa polypeptide would encode CNMT (1Choi K.B. Morishige T. Sato F. Phytochemistry. 2001; 56: 649-655Crossref PubMed Scopus (34) Google Scholar). Based on this observation, we determined the N-terminal amino acids, isolated the corresponding cDNA fragment, and finally the full-length cDNA. The nucleotide sequence of cDNA suggested that the deduced amino acid sequence showed some similarity to known NMTs such as phosphoethanolamine NMT. Further characterization of recombinant protein heterologously expressed in Escherichia coli confirmed that this isolated cDNA encoded CNMT. Recombinant CNMT was purified to homogeneity, and its enzymological properties were characterized. The evolution of CNMT involved in secondary metabolism is discussed based on the sequence similarity of this novel NMT. DISCUSSIONBased on the N-terminal amino acid sequence of purified CNMT and information on codon usage in Coptis genes, a full-length cDNA encoding CNMT was successfully cloned and its enzymatic activity was confirmed in a heterologous expression system. Recombinant CNMT purified from transgenic E. coli showed a significantly higher activity than that of CNMT purified from culturedCoptis cells. CNMT deduced from the cDNA sequence was 358 amino acids long with a calculated molecular mass of 41 kDa, which was slightly lower than the apparent molecular mass (45 kDa) of purified CNMT determined by SDS-PAGE. SDS-PAGE analysis of purified recombinant CNMT revealed a similar mobility for CNMT purified fromCoptis cells, indicating that this discrepancy between calculated molecular mass and apparent molecular mass was not due to post-translational modification but rather to experimental variation probably associated with the surface charge or conformation of CNMT.Because this CNMT sequence was the first reported NMT sequence in isoquinoline alkaloid biosynthesis and the third reported NMT in alkaloid biosynthesis in plants (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar, 14Kato M. Mizuno K. Crozier A. Fujimura T. Ashihara H. Nature. 2000; 406: 956-957Crossref PubMed Scopus (156) Google Scholar), the sequences of these plant alkaloid NMTs and other methyltransferases were compared to obtain more information about the diversity of methyltransferases. Whereas several plant O-methyltransferases have relatively high sequence homology ranging from 32 to 71%, the diversity ofN-methyltransferases is quite high (Fig. 3); the sequence identity among NMTs is about 5–15%. This means that each NMT may have evolved from an independent origin. CNMT showed relatively high homology to a hypothetical Arabidopsis protein (T05192) and cyclopropane-fatty acyl phospholipid synthase ofMesorhizobium (21Kaneko T. Nakamura Y. Sato S. Asamizu E. Kato T. Sasamoto S. Watanabe A. Idesawa K. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kiyokawa C. Kohara M. Matsumoto M. Matsuno A. Mochizuki Y. Nakayama S. Nakazaki N. Shimpo S. Sugimoto M. Takeuchi C. Yamada M. Tabata S. DNA Res. 2000; 7: 331-338Crossref PubMed Scopus (660) Google Scholar), whereas the functional similarity is not clear.The recent determination of the three-dimensional structures of plantO-methyltransferases suggested the structural conservation of an AdoMet binding site among methyltransferases (22Zubieta C. He X.Z. Dixon R.A. Noel J.P. Nat. Struct. Biol. 2001; 8: 271-279Crossref PubMed Scopus (271) Google Scholar). As Chandrashekhar and Vincent (23Chandrashekhar P.J. Vincent L.C. Plant Mol. Biol. 1998; 37: 663-674Crossref PubMed Scopus (234) Google Scholar) have deduced from 56 plant enzyme sequences, motif A (V/I/L)(V/L)(D/K)(V/I)GGXX(G/A) is present in all plant O-methyltransferases with 0–2 mismatches. Motifs B and C, (V/I/F)(A/P/E)X(A/P/G)DAXXXK(W/Y/F) and (A/P/G/S)(L/I/V)(A/P/G/S)XX(A/P/G/S)(K/R)(V/I)(E/I)(L/I/V) respectively, are conserved 98% with 0–3 mismatches (X is any amino acid). On the other hand, the AdoMet recognition domain was not as highly conserved in NMTs, even though motif A was found among almost all OMTs. In CNMT, only motif A is present with two mismatches (Fig. 6). The lower conservation of the AdoMet binding domain in NMT might be due to structural differences between NMTs and OMTs. Whereas many OMTs have an AdoMet binding site in their C-terminal half, motif A in CNMT and other NMTs is located at the N-terminal end (24Quaife C.J. Hoyle G.W. Froelick G.J. Findley S.D. Baetge E.E. Behringer R.R. Hammang J.P. Brinster R.L. Palmiter R.D. Transgenic Res. 1994; 3: 388-400Crossref PubMed Scopus (9) Google Scholar, 25Ying Z. Mulligan R.M. Janney N. Houtz R.L. J. Biol. Chem. 1999; 274: 36750-36756Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 26Scoilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (58) Google Scholar). Interestingly, a putativeArabidopsis protein (T05192) also has this motif A, and cyclopropane-fatty acyl phospholipid synthase also shows a similar identity in this region. Some enzymes in secondary metabolism such as putrescine N-methyltransferase and tropinone reductase have been postulated to have evolved from enzymes in primary metabolism such as spermine synthase or the short-chain dehydrogenase gene family (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar,27Nakajima K. Hashimoto T. Yamada Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9591-9595Crossref PubMed Scopus (118) Google Scholar). Further characterization of the enzyme activity ofArabidopsisT05192 and site-directed mutagenesis of CNMT should provide clues for understanding the evolution of CNMT. Based on the N-terminal amino acid sequence of purified CNMT and information on codon usage in Coptis genes, a full-length cDNA encoding CNMT was successfully cloned and its enzymatic activity was confirmed in a heterologous expression system. Recombinant CNMT purified from transgenic E. coli showed a significantly higher activity than that of CNMT purified from culturedCoptis cells. CNMT deduced from the cDNA sequence was 358 amino acids long with a calculated molecular mass of 41 kDa, which was slightly lower than the apparent molecular mass (45 kDa) of purified CNMT determined by SDS-PAGE. SDS-PAGE analysis of purified recombinant CNMT revealed a similar mobility for CNMT purified fromCoptis cells, indicating that this discrepancy between calculated molecular mass and apparent molecular mass was not due to post-translational modification but rather to experimental variation probably associated with the surface charge or conformation of CNMT. Because this CNMT sequence was the first reported NMT sequence in isoquinoline alkaloid biosynthesis and the third reported NMT in alkaloid biosynthesis in plants (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar, 14Kato M. Mizuno K. Crozier A. Fujimura T. Ashihara H. Nature. 2000; 406: 956-957Crossref PubMed Scopus (156) Google Scholar), the sequences of these plant alkaloid NMTs and other methyltransferases were compared to obtain more information about the diversity of methyltransferases. Whereas several plant O-methyltransferases have relatively high sequence homology ranging from 32 to 71%, the diversity ofN-methyltransferases is quite high (Fig. 3); the sequence identity among NMTs is about 5–15%. This means that each NMT may have evolved from an independent origin. CNMT showed relatively high homology to a hypothetical Arabidopsis protein (T05192) and cyclopropane-fatty acyl phospholipid synthase ofMesorhizobium (21Kaneko T. Nakamura Y. Sato S. Asamizu E. Kato T. Sasamoto S. Watanabe A. Idesawa K. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kiyokawa C. Kohara M. Matsumoto M. Matsuno A. Mochizuki Y. Nakayama S. Nakazaki N. Shimpo S. Sugimoto M. Takeuchi C. Yamada M. Tabata S. DNA Res. 2000; 7: 331-338Crossref PubMed Scopus (660) Google Scholar), whereas the functional similarity is not clear. The recent determination of the three-dimensional structures of plantO-methyltransferases suggested the structural conservation of an AdoMet binding site among methyltransferases (22Zubieta C. He X.Z. Dixon R.A. Noel J.P. Nat. Struct. Biol. 2001; 8: 271-279Crossref PubMed Scopus (271) Google Scholar). As Chandrashekhar and Vincent (23Chandrashekhar P.J. Vincent L.C. Plant Mol. Biol. 1998; 37: 663-674Crossref PubMed Scopus (234) Google Scholar) have deduced from 56 plant enzyme sequences, motif A (V/I/L)(V/L)(D/K)(V/I)GGXX(G/A) is present in all plant O-methyltransferases with 0–2 mismatches. Motifs B and C, (V/I/F)(A/P/E)X(A/P/G)DAXXXK(W/Y/F) and (A/P/G/S)(L/I/V)(A/P/G/S)XX(A/P/G/S)(K/R)(V/I)(E/I)(L/I/V) respectively, are conserved 98% with 0–3 mismatches (X is any amino acid). On the other hand, the AdoMet recognition domain was not as highly conserved in NMTs, even though motif A was found among almost all OMTs. In CNMT, only motif A is present with two mismatches (Fig. 6). The lower conservation of the AdoMet binding domain in NMT might be due to structural differences between NMTs and OMTs. Whereas many OMTs have an AdoMet binding site in their C-terminal half, motif A in CNMT and other NMTs is located at the N-terminal end (24Quaife C.J. Hoyle G.W. Froelick G.J. Findley S.D. Baetge E.E. Behringer R.R. Hammang J.P. Brinster R.L. Palmiter R.D. Transgenic Res. 1994; 3: 388-400Crossref PubMed Scopus (9) Google Scholar, 25Ying Z. Mulligan R.M. Janney N. Houtz R.L. J. Biol. Chem. 1999; 274: 36750-36756Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 26Scoilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (58) Google Scholar). Interestingly, a putativeArabidopsis protein (T05192) also has this motif A, and cyclopropane-fatty acyl phospholipid synthase also shows a similar identity in this region. Some enzymes in secondary metabolism such as putrescine N-methyltransferase and tropinone reductase have been postulated to have evolved from enzymes in primary metabolism such as spermine synthase or the short-chain dehydrogenase gene family (13Hibi N. Higashiguchi S. Hashimoto T. Yamada Y. Plant Cell. 1994; 6: 723-735Crossref PubMed Scopus (254) Google Scholar,27Nakajima K. Hashimoto T. Yamada Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9591-9595Crossref PubMed Scopus (118) Google Scholar). Further characterization of the enzyme activity ofArabidopsisT05192 and site-directed mutagenesis of CNMT should provide clues for understanding the evolution of CNMT. We thank Dr. Fang-Sik Che of Nara Institute of Science and Technology for N-terminal amino acid sequencing of CNMT. We also thank Dr. N. Nagakura and Mitsui PetroChemical Industries Ltd. for their generous gifts of the alkaloids. We thank Dr. W. Frommer of the University of Tübingen for the gift of pDR196." @default.
• W1991569676 created "2016-06-24" @default.
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• W1991569676 date "2002-01-01" @default.
• W1991569676 modified "2023-10-03" @default.
• W1991569676 title "Molecular Cloning and Characterization of CoclaurineN-Methyltransferase from Cultured Cells of Coptis japonica" @default.
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• W1991569676 doi "https://doi.org/10.1074/jbc.m106405200" @default.
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