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.
Highlights
Natural vitamin K has two molecular homologues: plant-derived vitamin K1, which contains a phytyl group side chain, and bacterial-derived vitamin K2, which contains a polyisoprenyl side chain
The microsomal fraction from UbiA prenyltransferase domain-containing protein 1 (UBIAD1)-expressing Sf9 cells synthesizes MK-4-d7 from MD-d8 using GGPP as an isoprenyl side-chain substrate, other isoprenyl side-chains are produced by the mevalonate pathway, including Geranyl diphosphate (GPP) and farnesyl pyrophosphate (FPP)
In order to eliminate the influence of endogenous coenzyme Q10 (CoQ10), 13C6-CoQ10 levels were analysed after the addition of 13C6-4HB; 13C6-CoQ10 levels were unchanged regardless of the amount of the UBIAD1 gene (S1 Fig)
Summary
Natural vitamin K has two molecular homologues: plant-derived vitamin K1 (phylloquinone: PK), which contains a phytyl group side chain, and bacterial-derived vitamin K2 (menaquinone-n: MK-n), which contains a polyisoprenyl side chain. Vitamin K is an essential cofactor required for γ-glutamyl carboxylase that converts specific glutamic acid residues into γ-carboxyglutamic acid residues in PLOS ONE | DOI:10.1371/journal.pone.0125737 April 15, 2015
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