title: Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1.
abstract: 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.
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan; Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan.
authors: Yoshihisa
authors: Y
authors: Hirota
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Kimie
authors: K
authors: Nakagawa
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Natsumi
authors: N
authors: Sawada
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Naoko
authors: N
authors: Okuda
authors: Department of Bioscience and Engineering, Shibaura Institute of Technology, Saitama, Japan.
authors: Yoshitomo
authors: Y
authors: Suhara
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Yuri
authors: Y
authors: Uchino
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Takashi
authors: T
authors: Kimoto
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Nobuaki
authors: N
authors: Funahashi
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Maya
authors: M
authors: Kamao
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Naoko
authors: N
authors: Tsugawa
authors: Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.
authors: Toshio
authors: T
authors: Okano
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053898:Biosynthetic Pathways; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004122:Dimethylallyltranstransferase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007313:Insecta; D008798:Mevalonic Acid / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D017368:Protein Prenylation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D024482:Vitamin K 2 / Q000378:metabolism*
chemical_list: D024482:Vitamin K 2; D002784:Cholesterol; D004122:Dimethylallyltranstransferase; C425464:UBIAD1 protein, human; D008798:Mevalonic Acid