Interaction between mitochondrial CYP27B1 and adrenodoxin: role of arginine 458 of mouse CYP27B1

A molecular modeling study of CYP27B1 suggests that Arg458 of mouse CYP27B1 is involved in interaction with adrenodoxin (ADX). Thus, we generated CYP27B1 mutants R458K and R458Q and revealed their enzymatic properties. Substrate-induced difference spectra and K(m) values for 1alpha-hydroxylation of 25(OH)D3 indicate that the replacement of Arg458 with Lys or Gln does not affect substrate binding. However, these mutants showed remarkable decreases of both kcat values and the ratio of product formation to NADPH oxidation (coupling efficiency). A high K(m) value of R458Q for ADX concentration and a decrease of rate constant of the first electron transfer seem reasonable considering that the conversion from Arg to noncharged Gln abolishes salt-bridge formation with the acidic residue of ADX. On the other hand, R458K showed atypical kinetics for ADX concentration with Hill's constant of 2.0 and high catalytic activity at high ADX concentration by increase of coupling efficiency. These results suggest that conformational change of R458K by binding the two ADX molecules is essential for 1alpha-hydroxylation of 25(OH)D3. On the other hand, binding one ADX molecule is sufficient for the conformational change of the wild-type CYP27B1, judging from its Michaelis-Menten-type kinetics for ADX concentration with high coupling efficiency. These results suggest that ADX functions as an effector for the oxygen transfer reaction in addition to being an electron donor for CYP27B1.     

Homology modeling of human 25-hydroxyvitamin D3 1alpha-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.     

Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)

A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.     

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 1alpha-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 1alpha-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.     

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.     

Conventional and tissue-specific inactivation of the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1)

Mutations in the human 25-hydroxyvitamin-D(3)-1alpha-hydroxylase (CYP27B1) gene cause pseudo vitamin D deficiency rickets (PDDR). The kidney is the main site of expression of the CYP27B1 gene, but expression has been documented in other cell types, including chondrocytes. We engineered a tissue-specific and a conventional knockout of CYP27B1 in mice. The conventional knockout strain reproduced the PDDR phenotype. Homozygote mutant animals were treated with 1,25(OH)(2)D(3) or fed a high-calcium diet (2% calcium, 1.25% phosphate, 20% lactose) for 5 weeks post-weaning. Blood biochemistry revealed that both rescue treatments corrected the hypocalcemia and secondary hyperparathyroidism. Bone histomorphometry confirmed that rickets were cured. The rescue regimen restored the biomechanical properties of the bone tissue. Mice carrying the loxP-bearing allele were bred to transgenic animals expressing the Cre recombinase in chondrocytes under the control of the collagen type II promoter. Genotyping confirmed excision of exon 8 in chondrocytes. Serum biochemistry revealed that mineral ion homeostasis is normal in mutant animals. Preliminary observation of bone tissue from mutant mice did not reveal major changes to the growth plate. Precise histomorphometric analysis will be required to assess the impact of chondrocyte-specific inactivation of CYP27B1 on the maturation and function of growth plate cells in vivo.     

CYP27B1 polymorphisms variants are associated with type 1 diabetes mellitus in Germans

CYP27B1 (25-hydroxyvitamin D(3)-1alpha-hydroxylase) catalyzes the metabolization of 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) the most active natural Vitamin D metabolite. 1,25(OH)(2)D(3) plays a role in the regulation of autoimmunity and cell proliferation and prevents the development of autoimmune diabetes mellitus in animal models besides other autoimmune disorders. One hundred and eighty-seven families with one offspring affected with type1diabetes mellitus were genotyped for the polymorphisms in the promoter region (-1260 C/A) and intron 6 (2338 T/C) of the CYP27B1 gene on chromosome 12 q13.1-13.3 and extended transmission disequilibrium tests (ETDT) were performed. The haplotype CT (-1260 A/2338 T) was significantly more often transmitted to affected offspring (96 transmitted (T) versus 63 not transmitted (NT), P = 0.0089). While the AT (-1260 C/2838 T) was significantly less often transmitted (37 T versus 60 NT, P = 0.0195). This study suggests that CYP27B1 haplotypes may confer susceptibility to type 1 diabetes mellitus in Germans.     

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.     

CYP2K6 from zebrafish (Danio rerio): cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme

A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.     

Influence of various polymorphic variants of cytochrome P450 oxidoreductase (POR) on drug metabolic activity of CYP3A4 and CYP2B6

Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ~70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.     

The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.     

CYP2R1-, CYP27B1- and CYP24-mRNA expression in German type 1 diabetes patients

1,25(OH)(2)D(3) and 25(OH)D(3) have been associated with type 1 diabetes. Diverse enzymes are involved in the synthesis of these metabolites: the 25-Vitamin-D-hydroxylase (CYP2R1), the 25-hydroxyvitamin-D(3)-1-alpha-hydroxylase (CYP27B1) and the 25(OH)D(3)-24-hydroxylase (CYP24) among others. Serum levels of 25(OH)D(3) and 1,25(OH)(2)D(3) were investigated in type 1 diabetes patients (n=173) and the mRNA expression of the CYP2R1, CYP27B1 and CYP24 genes in type 1 diabetes patients (n=33) and healthy controls (n=23). These parameters were correlated with the -1260 (C/A) polymorphism in the CYP27B1 gene. Lower expression of CYP27B1 mRNA in comparison with healthy controls (1.7165 versus 1.7815, P=0.0268) was found. Additionally, patients carrying the genotype CC possessed a reduced amount of CYP27B1 mRNA compared to healthy controls (1.6855 versus 1.8107, respectively, P=0.0220). The heterozygosity rate of the -1260 C/A polymorphism was more frequent in patients with normal levels of 1,25(OH)(2)D(3) (> or =19.9 pmol/ml) than in whose with a level of less than 19.9 pmol/ml (46.7% versus 22.2%, P=0.0134). No correlation with serum levels of 25(OH)D(3) was found. Thus, CYP27B1 gene could play a functional role in the pathogenesis of type 1 diabetes through modulation of its mRNA expression and influence serum levels of 1,25(OH)(2)D(3) via the -1260 C/A polymorphism.     

Characterization of Cupriavidus metallidurans CYP116B1--a thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein

The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus?metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 μM and K(m[NADH]) = 399.1 ± 52.1 μM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.