Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1

PLoS One. 2015 Apr 15;10(4):e0125737. doi: 10.1371/journal.pone.0125737. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biosynthetic Pathways
  • Cell Line
  • Cholesterol / metabolism
  • Dimethylallyltranstransferase / analysis
  • Dimethylallyltranstransferase / genetics
  • Dimethylallyltranstransferase / metabolism*
  • Gene Expression
  • Humans
  • Insecta
  • Mevalonic Acid / metabolism
  • Microsomes / enzymology
  • Microsomes / metabolism
  • Molecular Sequence Data
  • Point Mutation
  • Protein Prenylation
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Vitamin K 2 / metabolism*

Substances

  • Vitamin K 2
  • Cholesterol
  • Dimethylallyltranstransferase
  • TERE1 protein, human
  • Mevalonic Acid

Grant support

This work was supported in part by a Grant-in-aid for Scientific Research (B) (grant number 23390022 to TO: https://kaken.nii.ac.jp/d/r/20131542.en.html) from JSPS, a Grant-in-aid for Scientific Research (Young Scientists-B) (grant number 2379110 to KN: https://kaken.nii.ac.jp/d/r/90309435.en.html and 26860047 to YH: https://kaken.nii.ac.jp/d/r/70724277.en.html) from JSPS, and a Grant-in-aid for JSPS Fellows (grant number 24-7941 to YH: https://kaken.nii.ac.jp/d/p/12J07941.ja.html) from JSPS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.