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).

Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of ( S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)-Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.

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Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound, Kunitz-type serine protease inhibitor. HAI-1 inhibits serine proteases that have potent pro-hepatocyte growth factor-converting activity, such as the membrane-type serine protease, matriptase. HAI-1 comprises an N-terminal domain, followed by an internal domain, first protease inhibitory domain (Kunitz domain I), low-density lipoprotein receptor A module (LDLRA) domain, and a second Kunitz domain (Kunitz domain II) in the extracellular region. Our aim was to assess the roles of these domains in the inhibition of matriptase. Soluble forms of recombinant rat HAI-1 mutants made up with various combinations of domains were produced, and their inhibitory activities toward the hydrolysis of a chromogenic substrate were analyzed using a soluble recombinant rat matriptase. Kunitz domain I exhibited inhibitory activity against matriptase, but Kunitz domain II did not. The N-terminal domain and Kunitz domain II decreased the association rate between Kunitz domain I and matriptase, whereas the internal domain increased this rate. The LDLRA domain suppressed the dissociation of the Kunitz domain I-matriptase complex. Surprisingly, an HAI-1 mutant lacking the N-terminal domain and Kunitz domain II showed an inhibitor constant of 1.6 pM, and the inhibitory activity was 400 times higher in this HAI-1 mutant than in the mutant with all domains. These findings, together with the known occurrence of an HAI-1 species lacking the N-terminal domain and Kunitz domain II in vivo, suggest that the domain structure of HAI-1 is organized in a way that allows HAI-1 to flexibly control matriptase activity. Hepatocyte growth factor (HGF)3 activator inhibitor type 1 (HAI-1) is an epithelial-derived, serine protease inhibitor with multiple domains, including two protease-inhibiting Kunitz domains (1, 2). HAI-1 was isolated originally from the conditioned medium of a human stomach carcinoma MKN45 cell line as a potent inhibitor of HGF activator, a 53-kDa serine protease responsible for proteolytic activation of the inactive single chain precursor of HGF (pro-HGF) (1, 3). HAI-1 is believed to play a crucial role in growth factor-mediated biological processes such as tissue regeneration by counteracting the HGF activator activity (2, 4).The primary translation product of HAI-1 predicted from the cDNA sequence comprises 513 amino acid residues, including a putative N-terminal signal peptide sequence and a hydrophobic region at the C-terminal region (Fig. 1A). This suggests that HAI-1 is produced first as a type I membrane protein (1). Indeed, the transmembrane form of HAI-1, which has a molecular mass of 66 kDa, was detected in extracts of MKN45 cells (5) and monkey kidney COS-1 cells transiently transfected with a rat HAI-1 cDNA (6). The extracellular domain can be released by cleavage with certain proteases (5). At least two HAI-1 species of 58 and 40 kDa are found in conditioned media of MKN45 cells (1, 5) and transfected COS-1 cells (6). The 58-kDa species (58-kD...

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...