Natsumi Sawada

Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme

Vitamin K occurs in the natural world in several forms, including a plant form, phylloquinone (PK), and a bacterial form, menaquinones (MKs). In many species, including humans, PK is a minor constituent of hepatic vitamin K content, with most hepatic vitamin K content comprising long-chain MKs. Menaquinone-4 (MK-4) is ubiquitously present in extrahepatic tissues, with particularly high concentrations in the brain, kidney and pancreas of humans and rats. It has consistently been shown that PK is endogenously converted to MK-4 (refs 4–8). This occurs either directly within certain tissues or by interconversion to menadione (K3), followed by prenylation to MK-4 (refs 9–12). No previous study has sought to identify the human enzyme responsible for MK-4 biosynthesis. Previously we provided evidence for the conversion of PK and K3 into MK-4 in mouse cerebra. However, the molecular mechanisms for these conversion reactions are unclear. Here we identify a human MK-4 biosynthetic enzyme. We screened the human genome database for prenylation enzymes and found UbiA prenyltransferase containing 1 (UBIAD1), a human homologue of Escherichia coli prenyltransferase menA. We found that short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin K derivatives into deuterium-labelled-MK-4 (MK-4-d7) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin K derivatives into MK-4-d7 in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d7 was chemically identified by 2H-NMR analysis. MK-4 biosynthesis by UBIAD1 was not affected by the vitamin K antagonist warfarin. UBIAD1 was localized in endoplasmic reticulum and ubiquitously expressed in several tissues of mice. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme; this identification will permit more effective decisions to be made about vitamin K intake and bone health.

Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1.

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1α-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1α-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.

Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats.

Background: Menadione is an intermediate in phylloquinone to menaquinone-4 conversion in mammals. Results: Menadione is released from phylloquinone in the intestine and converted to menaquinone-4 in tissues after being reduced. Conclusion: Menadione is a catabolic product of phylloquinone and circulating precursor of tissue menaquinone-4. Significance: Determining how phylloquinone is metabolized in the body is crucial for understanding vitamin K biology. Mice have the ability to convert dietary phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K3), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and 1H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.

Cell specificity and properties of the C-3 epimerization of Vitamin D3 metabolites

It is well documented that Vitamin D3 metabolites and synthetic analogs are metabolized to their epimers of the hydroxyl group at C-3 of the A-ring. We investigated the C-3 epimerization of Vitamin D3 metabolites in various cultured cells and basic properties of the enzyme responsible for the C-3 epimerization. 1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3], 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were metabolized to the respective C-3 epimers in UMR-106 (rat osteosarcoma), MG-63 (human osteosarcoma), Caco-2 (human colon adenocarcinoma), LLC-PK1 (porcine kidney) and HepG2 (human hepatoblastoma)] cells, although the differences existed in the amount of each C-3 epimer formed with different cell types. In terms of maximum velocity (Vmax) and Michaelis constant (Km) values for the C-3 epimerization in microsome fraction of UMR-106 cells, 25(OH)D3 exhibited the highest specificity for the C-3 epimerization among 1alpha,25(OH)2D3, 25(OH)D3 and 24,25(OH)2D3. C-3 epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha --> beta) -hydroxysteroid epimerase (HSE) catalyzed the C-3 epimerization in vitro. Based on these results, the enzyme responsible for the C-3 epimerization of Vitamin D3 are thought to be different from already-known cytochrome P450-related Vitamin D metabolic enzymes and HSE.

Homology modeling of human 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1) based on the crystal structure of rabbit CYP2C5 ☆

Seventeen missense mutations of 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) that cause Vitamin D-dependent rickets type I (VDDR-I) have been identified. To understand the mechanism by which each mutation disrupts 1alpha-hydroxylase activity and to visualize the substrate-binding site, we performed the homology modeling of CYP27B1. The three-dimensional (3D) structure of CYP27B1 was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of a eukaryotic CYP. The 3D structure of CYP27B1 contains 17 helices and 6 beta-strands, and the overall structural folding is similar to the available structures of soluble CYPs as well as to the template CYP2C5. Mapping of the residues responsible for VDDR-I has provided much information concerning the function of each mutant. We have previously reported site-directed mutagenesis studies on several mutants of CYP27B1 causing VDDR-1, and suggested the role of each residue. All these suggestions are in good agreement with our 3D-model of CYP27B1. Furthermore, this model enabled us to predict the function of the other mutation residues responsible for VDDR-I.

Metabolism of 26,26,26,27,27,27-F6-1α,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3). The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3). The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3). In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24. Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway. Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.

Functional Characterization of the Vitamin K2 Biosynthetic Enzyme UBIAD1

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.

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1 −/−) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1 −/− embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1 +/− mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1 +/− E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1 −/− mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1 +/− mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

Overexpression of RGPR-p117 enhances regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells: involvement of TTGGC motif.

A novel protein RGPR‐p117 was discovered as regucalcin gene promoter region‐related protein that binds to the TTGGC motif using a yeast one‐hybrid system. RGPR‐p117 is localized in the nucleus of kidney cells, and overexpression of RGPR‐p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR‐p117 enhances the regucalcin promoter activity using the −710/+18 LUC construct (wild‐type) or −710/+18 LUC construct (mutant) with deletion of −523/−435 including TTGGC motif. NRK52E cells (wild‐type) or stable HA‐RGPR‐p117/phCMV2‐transfected cells (transfectant) were cultured in Dulbeccos minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild‐type cells or transfectants were transfected with the −710/+18 LUC construct vector or the −710/+18 LUC construct with deletion of −523/−435. Wild‐type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1–34) (PTH; 10−7 M). Luciferase activity in wild‐type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild‐type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of −523/−435 sequence of regucalcin promoter. This was also seen using the −710/+18 LUC construct with deletion of −523/−503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10−7 M), Bay K 8644 (10−6 M), phorbol 12‐myristate 13‐acetate (PMA; 10−6 M), or N6, 2′‐dibutyryl cyclic adenosine 3′, 5′‐monophosphate (DcAMP; 10−4 M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10−6 M), staurosporine (10−9 M), PD 98059 (10−8 M), wortmannin (10−8 M), genistein (10−6 M), vanadate (10−6 M), or okadaic acid (10−6 M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR‐p117 can enhance the regucalcin promoter activity which is related to the NF‐1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells. J. Cell. Biochem. 99: 589–597, 2006.

Replacement of the hydrophobic part of 9-cis-retinoic acid with cyclic terpenoid moiety results in RXR-selective agonistic activity

Retinoid X receptor (RXR) agonists are interesting candidates for the treatment of metabolic syndrome. 9-Cis-retinoic acid (9cRA: 1) is a natural RXR agonist, that also works as a retinoic acid receptor (RAR) agonist. This fact prompted us to study the structure-activity relationship (SAR) of RXR agonists derived from 1. Though 3 and 4, in which the cyclohexene part of 1 is replaced with bulkier hydrophobic moieties, show RXR-selective agonistic activity, some analogs containing other ring structures show RAR agonistic activity. Thus, we were interested in establishing what kind of ring skeleton is required for RXR-selective agonistic activity. In this study, we systematically prepared 5 and 6, in which the cyclohexene ring of 1 is replaced with various cyclic terpenoid moieties, and evaluated their RXR and RAR agonistic activities. Our previously reported CsF-promoted Stille coupling reaction was employed as a key step for the comprehensive synthesis of 5 and 6. The results of transcriptional assay showed that compounds 5b-f, which possess a menthane skeleton, exhibit RXR-selective agonistic activity. These results should be helpful for the design of superior RXR-selective agonists based on the structure of 1.