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<p>We have cloned cDNA encoding an <i>S</i>-adenosyl-<span>l</span>-methionine (SAM)-dependent cinnamic acid carboxyl methyltransferase from the basidiomycete <i>Tricholoma matsutake</i>. For cloning, we focused on four conserved motifs in SABATH (<Emphasis Type="Underline">S</Emphasis>alicylic <Emphasis Type="Underline">A</Emphasis>cid, <Emphasis Type="Underline">B</Emphasis>enzoic <Emphasis Type="Underline">A</Emphasis>cid and <Emphasis Type="Underline">TH</Emphasis>eobromine)-family proteins, including the cinnamic acid carboxyl methyltransferases from <i>Ocimum basillicum</i> (ObCCMT1) and <i>Conocephalum salebrosum</i> (CsCAMT), not their full amino acid sequences. On the basis of the homology of the motifs between ObCCMT1 and putative SAM-dependent methyltransferases predicted in RNA-Seq data for <i>T. matsutake</i>, we selected and cloned three <i>T. matsutake</i> cDNAs, named <i>1391973</i>, <i>1389993</i>, and <i>1330427</i>, respectively. Recombinant 1330427 protein showed the target activity, and hence was renamed TmCAMT1 (<Emphasis Type="ItalicUnderline">T</Emphasis><i>. </i><Emphasis Type="ItalicUnderline">m</Emphasis><i>atsutake</i> <Emphasis Type="Underline">C</Emphasis>innamic <Emphasis Type="Underline">A</Emphasis>cid carboxyl <Emphasis Type="Underline">M</Emphasis>ethyl<Emphasis Type="Underline">T</Emphasis>ransferase). The identities between TmCAMT1 and ObCCMT1 and CsCAMT were just 17% and 19%, respectively. TmCAMT1 was categorized into Pfam database family Methyltransf_31, not Methyltransf_7 to which ObCCMT1 and CsCAMT belong. However, structural prediction using AlphaFold2 and comparison with the crystal structure of <i>Clarkia breweri</i> SAM-dependent salicylic acid carboxyl methyltransferase (CbSAMT; PDB ID: 1M6E) indicated that TmCAMT1, ObCCMT1, CsCAMT and CbSAMT are similar in overall structure and amino acid residues in the active site. Meanwhile, during the course of this study, MnMT2, a cinnamic acid carboxyl methyltransferase from mycobacteria, was reported. TmCAMT1 shows slightly greater identity, 21%, with MnMT2, although MnMT2 belongs to the leucine carboxyl methyltransferase family, not the SABATH family, and AlphaFold2 predicted that TmCAMT1 and MnMT2 have limited overall structural similarity. In conclusion, our cloning strategy successfully obtained TmCAMT1, the first characterized SAM-dependent cinnamic acid carboxyl methyltransferase that is similar to SABATH family proteins in overall structure and contains a few motifs of this family, but it comes from a non-plant species. However, the low <i>k</i>cat value suggests that further research is required to elucidate whether TmCAMT1 plays a predominant role for (<i>E</i>)-methyl cinnamate biosynthesis in <i>T. matsutake</i>.</p>

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S-Adenosyl-l-methionine-dependent cinnamic acid carboxyl methyltransferase from the basidiomycete Tricholoma matsutake, which has low identity but similar overall structure to plant SABATH-family enzymes

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  • 发表日期：

    2025年3月15日

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  • Takefumi Hattori,
  • Megumi Katayama,
  • Hiromitsu Tsuzuki,
  • Yumi Okamoto,
  • Kyosuke Ida,
  • Mariko Yoshizumi,
  • Masanori Abe,
  • Ryushi Kawakami,
  • Junji Hayashi,
  • Masaomi Yamamura

摘要

We have cloned cDNA encoding an S-adenosyl-l-methionine (SAM)-dependent cinnamic acid carboxyl methyltransferase from the basidiomycete Tricholoma matsutake. For cloning, we focused on four conserved motifs in SABATH (Salicylic Acid, Benzoic Acid and THeobromine)-family proteins, including the cinnamic acid carboxyl methyltransferases from Ocimum basillicum (ObCCMT1) and Conocephalum salebrosum (CsCAMT), not their full amino acid sequences. On the basis of the homology of the motifs between ObCCMT1 and putative SAM-dependent methyltransferases predicted in RNA-Seq data for T. matsutake, we selected and cloned three T. matsutake cDNAs, named 1391973, 1389993, and 1330427, respectively. Recombinant 1330427 protein showed the target activity, and hence was renamed TmCAMT1 (T. matsutake Cinnamic Acid carboxyl MethylTransferase). The identities between TmCAMT1 and ObCCMT1 and CsCAMT were just 17% and 19%, respectively. TmCAMT1 was categorized into Pfam database family Methyltransf_31, not Methyltransf_7 to which ObCCMT1 and CsCAMT belong. However, structural prediction using AlphaFold2 and comparison with the crystal structure of Clarkia breweri SAM-dependent salicylic acid carboxyl methyltransferase (CbSAMT; PDB ID: 1M6E) indicated that TmCAMT1, ObCCMT1, CsCAMT and CbSAMT are similar in overall structure and amino acid residues in the active site. Meanwhile, during the course of this study, MnMT2, a cinnamic acid carboxyl methyltransferase from mycobacteria, was reported. TmCAMT1 shows slightly greater identity, 21%, with MnMT2, although MnMT2 belongs to the leucine carboxyl methyltransferase family, not the SABATH family, and AlphaFold2 predicted that TmCAMT1 and MnMT2 have limited overall structural similarity. In conclusion, our cloning strategy successfully obtained TmCAMT1, the first characterized SAM-dependent cinnamic acid carboxyl methyltransferase that is similar to SABATH family proteins in overall structure and contains a few motifs of this family, but it comes from a non-plant species. However, the low kcat value suggests that further research is required to elucidate whether TmCAMT1 plays a predominant role for (E)-methyl cinnamate biosynthesis in T. matsutake.