Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1

Yoshihisa Hirota, Kimie Nakagawa, Natsumi Sawada, Naoko Okuda, Yoshitomo Suhara, Yuri Uchino, Takashi Kimoto, Nobuaki Funahashi, Maya Kamao, Naoko Tsugawa, Toshio Okano

研究成果: Article

23 引用 (Scopus)

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UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.

元の言語English
記事番号e0125737
ジャーナルPLoS ONE
10
発行部数4
DOI
出版物ステータスPublished - 2015 4 15

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

フィンガープリント Functional characterization of the vitamin K<sub>2</sub> biosynthetic enzyme UBIAD1' の研究トピックを掘り下げます。これらはともに一意のフィンガープリントを構成します。

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    Hirota, Y., Nakagawa, K., Sawada, N., Okuda, N., Suhara, Y., Uchino, Y., Kimoto, T., Funahashi, N., Kamao, M., Tsugawa, N., & Okano, T. (2015). Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1. PLoS ONE, 10(4), [e0125737]. https://doi.org/10.1371/journal.pone.0125737