Characterization of Class IB Terpene Synthase: The First Crystal Structure Bound with a Substrate Surrogate

Rafaella Stepanova, Hayato Inagi, Kei Sugawara, Kazuya Asada, Tomoyuki Nishi, Daijiro Ueda, Yoko Yasuno, Tetsuro Shinada, Kunio Miki, Masahiro Fujihashi, Tsutomu Sato

研究成果: Contribution to journalArticle

抜粋

Terpene synthases (TS) are classified into two broad types, Class I and II, based on the chemical strategy for initial carbocation formation and motif sequences of the catalytic site. We have recently identified a new class of enzymes, Class IB, showing the acceptability of long (C20-C35) prenyl-diphosphates as substrates and no amino acid sequence homology with known TS. Conversion of long prenyl-diphosphates such as heptaprenyl-diphosphate (C35) is unusual and has never been reported for Class I and II enzymes. Therefore, the characterization of Class IB enzymes is crucial to understand the reaction mechanism of the extensive terpene synthesis. Here, we report the crystal structure bound with a substrate surrogate and biochemical analysis of a Class IB TS, using the enzyme from Bacillus alcalophilus (BalTS). The structure analysis revealed that the diphosphate part of the substrate is located around the two characteristic Asp-rich motifs, and the hydrophobic tail is accommodated in a unique hydrophobic long tunnel, where the C35 prenyl-diphosphate, the longest substrate of BalTS, can be accepted. Biochemical analyses of BalTS showed that the enzymatic property, such as Mg2+ dependency, is similar to those of Class I enzymes. In addition, a new cyclic terpene was identified from BalTS reaction products. Mutational analysis revealed that five of the six Asp residues in the Asp-rich motifs and two His residues are essential for the formation of the cyclic skeleton. These results provided a clue to consider the application of the unusual large terpene synthesis by Class IB enzymes.

元の言語英語
ページ(範囲)1517-1525
ページ数9
ジャーナルACS Chemical Biology
15
発行部数6
DOI
出版物ステータス出版済み - 6 19 2020
外部発表Yes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine

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    Stepanova, R., Inagi, H., Sugawara, K., Asada, K., Nishi, T., Ueda, D., Yasuno, Y., Shinada, T., Miki, K., Fujihashi, M., & Sato, T. (2020). Characterization of Class IB Terpene Synthase: The First Crystal Structure Bound with a Substrate Surrogate. ACS Chemical Biology, 15(6), 1517-1525. https://doi.org/10.1021/acschembio.0c00145