title: Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B' methyltransferase family in Coffea arabica.
abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-(14)C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B'-MTs. It was concluded that CTgSs have strict substrate specificity. The K(m) values of CTgS1 and CTgS2 were 121 and 184μM with nicotinic acid as a substrate, and 68 and 120μM with S-adenosyl-L-methionine as a substrate, respectively.
authors.affiliation: Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan. Electronic address: koumno@akita-pu.ac.jp.
authors.forename: Kouichi
authors.initials: K
authors.lastname: Mizuno
authors.affiliation: Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Matsuzaki
authors.affiliation: Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Kanazawa
authors.affiliation: Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Tokiwano
authors.affiliation: Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Yoshizawa
authors.affiliation: Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
authors.forename: Misako
authors.initials: M
authors.lastname: Kato
mesh_terms: D000470:Alkaloids / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D040503:Coffea / Q000201:enzymology*; D004789:Enzyme Activation; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009525:Niacin / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000470:Alkaloids; D009525:Niacin; C009560:trigonelline; D008780:Methyltransferases; C120479:caffeine synthase
keywords: Coffee; N-methyltransferase; Nicotinic acid; S-adenosylmethionine; Trigonelline