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).

Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.Although the brain is the most cholesterol-rich organ in the body, relatively little is known about the mechanisms by which it maintains steady-state cholesterol levels (1, 2). This is in marked contrast to the situation in virtually every other tissue or organ. One finding that has been consistently confirmed is that, due to the efficiency of the bloodbrain barrier, the brain is unable to take up cholesterol from the circulation and relies on de novo synthesis to meet its substantial cholesterol requirements. However, the rate of cholesterol synthesis in the adult brain is very low, and the bulk of brain cholesterol has a half-life that is at least 100 times longer than that of cholesterol in most other organs (3).One consequence of this "uncoupling" of brain and whole body cholesterol homeostasis has been the evolution of specific mechanisms for maintenance of cerebral cholesterol levels. Two mechanisms for removal of brain cholesterol are currently recognized (1). The first is analogous to classic "reverse cholesterol transport" and is mediated by a flux of cholesterol present in apolipoprotein E containing lipoproteins through cerebrospinal fluid into the circulation (4, 5). In adults, this mechanism is believed to be responsible for elimination of 1-2 mg of cholesterol per 24 h. The details of this particular ...

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In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.