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.

Fig 1. Human UBIAD1 Prenyltransferase Activity.

(A) Effect of DTT on prenyltransferase activity of human UbiA prenyltransferase domain-containing protein 1 (UBIAD1). (B) pH dependence of prenyltransferase activity of human UBIAD1. Grey circles indicate the PIPES buffer (pH 6.0–7.5; 100 mM), black circles the Tris buffer (pH 7.0–9.5; 100 mM) and white circles indicate the CHES buffer (pH 9.0–10.5; 100 mM). (C) Magnesium dependence of prenyltransferase activities of human UBIAD1. Three kinds of prenyldiphosphates, GPP (grey bar), farnesyl pyrophosphate (FPP) (white bar) and GGPP (black bar), were used as substrates. Significantly different from cells treated with 0-mM MgCl₂; ***P < 0.001, Dunnett’s test. N.D.: not detected. (D) Effect of various divalent metal ions on prenyltransferase activities of human UBIAD1. Significantly different from non-treated cells; ***P < 0.001, Dunnett’s test. N.D.: not detected.

Fig 2. Influence of Statins and Bisphosphonates on Human UbiA Prenyltransferase Domain-containing Protein 1 Activity.

(A) Effect of statins on MK-4 conversion activity in MG63 cells. (B) Effect of bisphosphonates on MK-4 conversion activity in MG63 cells. Values not sharing a common letter in each group are significantly different (Tukey–Kramer HSD-test): P < 0.05. Significantly different from MD-d₈-treated cells; *P < 0.05, Dunnett’s test. N.D.: not detected. (C) Geranylgeranyl diphosphate (GGPP) dose-dependent MK-4 conversion activity of UBIAD1 in statin-treated MG63 cells. (D) GGPP dose-dependent MK-4 conversion activity of UBIAD1 in bisphosphonate-treated MG63 cells. Values not sharing a common letter in each group are significantly different (Tukey–Kramer HSD-test): P < 0.05. N.D.: not detected. (E) Statin dependence of prenyltransferase activities of UBIAD1 in Sf9-UBIAD1 microsomes. Significantly different from non-treated cells; *P < 0.05, ***P < 0.001, Dunnett’s test. (F) Bisphosphonates dependence of prenyltransferase activities of UBIAD1 in Sf9-UBIAD1 microsomes.

Fig 3. Representative Features of the Human UBIAD1 Protein and Multiple Alignment of Regions Conserved among Prenylation Enzyme Family Members involved in Menaquinone and Coenzyme Q Synthesis.

(A) Highly concerved region of human UbiA prenyltransferase domain-containing protein 1 (UBIAD1). Gray box indicates the highly concerved region. (B) Phylogenetic analysis of the UbiA prenyltransferase family. (C) Amino acid residues corresponding to Regions I, II, III and IV. (D) Immunoblotting of human UBIAD1 expressed in Sf9 cells microsomal proteins. The UBIAD1 band size is 36.8 kDa and the GAPDH band size is 36.5 kDa. WT:wild type.

Fig 4. MK-n Synthesis Activity of Human UBIAD1 Mutants of Conserved Aminoacids.

(A) Schematic representation the prenylation mechanisms of MD (MD-d₈) conversion to menaquinones (MK-n-d₇). (B) MK-n synthetic activity of human UbiA prenyltransferase domain-containing protein 1 (UBIAD1) deletion mutants. Significantly different from wild type (WT) UBIAD1; ***P < 0.001, Dunnett’s test. N.D.: not detected. (C) MK-n synthetic activity of human UBIAD1 point mutants by alanine scanning. Significantly different from wild type UBIAD1; *P < 0.05, **P < 0.01, ***P < 0.001, Dunnett’s test. N.D.: not detected. (D) MK-n synthetic activity of human UBIAD1 point mutants caused by Schnyder corneal dystrophy. Significantly different from wild type UBIAD1; *P < 0.05, **P < 0.01, ***P < 0.001, Dunnett’s test. N.D.: not detected.

Fig 5. MK-4 Synthetic Activity and Cholesterol Biosynthesis Activities of Human UBIAD1 Point Mutants by Alanine Scanning in MG63 Cells.

(A) mRNA expression in human UbiA prenyltransferase domain-containing protein 1 (UBIAD1) point mutants by alanine scanning. Significantly different from control; ***P < 0.001, Dunnett’s test. WT:wild type. (B) MK-4 synthetic activity of human UBIAD1 point mutants by alanine scanning. Significantly different from control; **P < 0.01, ***P < 0.001, Dunnett’s test. Significantly different from wild type; ^###P < 0.001, Student’s t-test. (C) Cholesterol biosynthetic activity of human UBIAD1 point mutants by alanine scanning. Significantly different from the control; *P < 0.05, **P < 0.01, Student’s t test.

Fig 6. Subcellular Localization of Human UBIAD1 in MG63 cells.

MG63 cells were transfected with pEBMulti-UBIAD1 mutant. Cells were then stained with UBIAD1 antibody (Green) and the Golgi apparatus was stained with GOLGA5 (Red). As a control, we used the empty vector pEBMulti (Control). Cells were viewed under a LSM 700 microscope and photographed. All experiments were repeated at least three times. White arrows indicate areas with both green and red staining. WT: wild type.