Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1
| Autor: | Natsumi Sawada, Toshio Okano, Yuri Uchino, Naoko Okuda, Naoko Tsugawa, Maya Kamao, Yoshihisa Hirota, Kimie Nakagawa, Yoshitomo Suhara, Nobuaki Funahashi, Takashi Kimoto |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Insecta
Molecular Sequence Data Protein domain Protein Prenylation Gene Expression Mevalonic Acid lcsh:Medicine Sequence alignment Biology Cell Line chemistry.chemical_compound Prenylation EVH1 domain Dimethylallyltranstransferase Microsomes Animals Humans Point Mutation Amino Acid Sequence Binding site lcsh:Science Multidisciplinary Geranyl pyrophosphate lcsh:R Vitamin K 2 Biosynthetic Pathways Protein Structure Tertiary Cholesterol Biochemistry chemistry Protein prenylation lcsh:Q Sequence Alignment Research Article |
| Zdroj: | PLoS ONE, Vol 10, Iss 4, p e0125737 (2015) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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