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 (K(3)), 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 K(3) 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-d(7)) 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-d(7) in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d(7) was chemically identified by (2)H-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.

Human 25-hydroxyvitamin D 3 (25(OH)D 3 ) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D 3 , 1a,25(OH) 2 D 3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D 3 and 1a,25(OH) 2 D 3 , whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada . We also succeeded in the coexpression of CYP24, adrenodoxin and NADPHadrenodoxin reductase in E. coli. Addition of 25(OH)D 3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. . The complicated metabolic pathways, including . 30 metabolites [7], suggested that many enzymes were related to the metabolism. However, our recent study on rat CYP24 [8] revealed that at least six-step monooxygenation toward 1a,25(OH) 2 D 3 and four-step monooxygenation toward 25(OH)D 3 could be catalyzed by rat CYP24. Although rat CYP24 showed only C-24 hydroxylation pathway, human CYP24 was reported to catalyze 23S-hydroxylation of 25(OH)D 3 [9] which is the first step in the C-23 hydroxylation pathway. In this paper, we report the further metabolism of 25(OH)D 3 to 25(OH)D 3 -26,23-lactone in C-23 hydroxylation pathway by human CYP24. Remarkable metabolism towards 25(OH)D 3 and 1a,25(OH) 2 D 3 by human CYP24 are demonstrated.Vitamin D analogs are potentially useful in the clinical treatment of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia and breast cancer [7]. The metabolism of vitamin D analogs in target tissues such as kidney, small intestine and bones is pharmacologically essential as reported by Komuro et al. [10]. The major metabolic enzyme of the vitamin D analogs in these tissues is considered to be CYP24 [10,11]. Species differences observed in the metabolism of these vitamin D 3 analogs appear to originate from the specificity of CYP24-dependent reactions. Because human kidney specimens are not obtained easily, an in vitro system containing human CYP24 is required to predict drug metabolism in the human kidney. Here, we show the overexpression of human CYP24 in Escherichia coli. The expression level of CYP24 appears to be much higher than that in Sf21 cells using a baculovirus system as reported by Beckman et al. [9]. As Eur. J. Biochem. 267, 6158±6165 (2000) [25][26]24,25,26, ; tetranor 1a,23(OH) 2 , 24,25,26,27-tetranor-1a,23-dihydroxyvitamin D 3 ; tetranor 23(OH), 24,25,26,27-tetranor-23-hydroxyvitamin D 3 . Enzyme: bovine NADPH-adrenodoxin reductase (EC 1.18.1.2).

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.

Previously we expressed rat 25-hydroxyvitamin D 3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D 3 and 1a, 25-dihydroxyvitamin D 3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335±343]. In this study, we demonstrate further oxidation by CYP24 including four-and six-step monooxygenation towards 25-hydroxyvitamin D 3 and 1a,25-dihydroxyvitamin D 3 , respectively. When the substrate 25-hydroxyvitamin D 3 was added to a culture of recombinant E. coli, four metabolites, 24,25-dihydroxyvitamin D 3 , 24-oxo-25-hydroxyvitamin D 3 , 24-oxo-23,25-dihydroxyvitamin D 3 and 24,25,26,27-tetranor-23-hydroxyvitamin D 3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D 3 . Furthermore, in-vivo and in-vitro metabolic studies on 1a,25-dihydroxyvitamin D 3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1a,25-dihydroxyvitamin D 3 into calcitroic acid which is known as a final metabolite of 1a,25-dihydroxyvitamin D 3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D 3 and 1a,25-dihydroxyvitamin D 3 .Keywords: CYP24; electron transfer; P450, vitamin D.During the last decade, many mammalian P450 species have been expressed in Escherichia coli cells [1±4] mainly for the purpose of overproduction of the P450s. A merit of the E. coli expression system is the low background as compared with eukaryotic expression systems; this allows characterization of the expressed P450. Complete genome sequence analysis of E. coli K12 suggested the absence of a P450 gene in the genome [5] and E. coli has no steroids in the cell membranes; these facts strongly suggest that the E. coli expression system is useful for enzymatic studies of steroidogenic P450s.Barnes et al.[2] reported the interesting finding that mammalian microsomal P450 can exhibit monooxygenase activity in E. coli cells. Electrons are transferred from NADPH through NADPH-flavodoxin reductase and flavodoxin to microsomal P450s. NADPH-flavodoxin reductase and flavodoxin contain a flavin adenine dinucleotide (FAD) and a flavin mononucleotide (FMN) molecule, respectively. Thus, these two enzymes function as an electron transfer system instead of a mammalian microsomal NADPH-P450 reductase which contains both FAD and FMN molecules. However, on mitochondrial P450s such as P450scc (CYP11A) [4] and P450c27 (CYP27) [6] no report showing monooxygenase activity in living E. coli cells has been published. In this report, we describe the presence of an electron transfer system for the mitochondrial P450s in E. coli cells.Previous studies in vitro using the membrane fraction of recombinant E. coli cells indicated that rat P450c24 (CYP24) is not only active in 24-hydroxylation but is also responsible for the subsequent two hydroxylation steps in the metabolis...

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1, 2). 1␣,25(OH) 2 D 3 has potent anti-proliferative and cell differentiation-inducing activities in addition to its role in calcium homeostasis. After the expression of various biological activities, 1␣,25(OH) 2 D 3 is further metabolized through the C-24 (3-6)/C-23 (7-10) oxidation pathways and the C-3 epimerization pathway (11-14). The C-24 oxidation pathway, initiated by C-24 hydroxylation, leads to the conversion of 1␣,25(OH) 2 D 3 into a side chain cleavage product, calcitroic acid (4, 5). The C-23 oxidation pathway, initiated by C-23 hydroxylation, leads to the formation of 1␣,25(OH) 2 D 3 -26,23-lactone (7-10). The newly discovered C-3 epimerization pathway leads to the conversion of the configuration of the hydroxyl group at C-3 of the A-ring and produces 3-epi-1␣,25(OH) 2 D 3 from 1␣,25(OH) 2 D 3 . In view of this modification at the A-ring, the C-3 epimerization pathway is quite different from side chain oxidation pathways.The C-3 epimerization of 1␣,25(OH) 2 D 3 was observed in human colon carcinoma-derived Caco-2 cells (11), bovine parathyroid cells (12), rat osteoblastic UMR 106 and Ros17/2.8 cells (13), and various cultured cell lines (14). From these studies, the C-3 epimerization pathway is assumed to be cell-selective. It was considered that the C-3 epimerization pathway is cell differentiation-related in Caco-2 cells, because 3-epi-1␣,25 (OH) 2 D 3 was only observed in confluent, quiescent Caco-2 cells, not proliferating Caco-2 cells (11). 3-Epi-1␣,25(OH) 2 D 3 was also isolated as a circulating metabolite of 1␣,25(OH) 2 D 3 in rats treated with pharmacological doses of 1␣,25(OH) 2 D 3 (15). In addition, synthetic analogs of 1␣,25(OH) 2 D 3 , e.g. 22-oxacalcitriol (16), 20-epi-1␣,25(OH) 2 D 3 (17), and 1␣,25(OH) 2 -16-ene-23-yne-D 3 (18), have been reported to be metabolized to their * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, a grant for Cooperative Research administered by the Japan Private School Promotion Foundation, and a grant-in-aid from the Ministry of Health and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ § To whom correspondence should be addressed: Dept.

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.

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