Three-step hydroxylation of vitamin D 3 by a genetically engineered CYP105A1 Enzymes and catalysis Keiko Hayashi 1 , Kaori Yasuda 1 , Hiroshi Sugimoto 2 , Shinichi Ikushiro 1 , Masaki Kamakura 1 , Atsushi Kittaka 3 , Ronald L. Horst 4 , Tai C. Chen 5 , Miho Ohta 6 , Yoshitsugu Shiro 2 and Toshiyuki Sakaki 1 1 Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Kurokawa, Imizu, Toyama, Japan 2 RIKEN SPring-8 Center, Harima Institute, Sayo, Hyogo, Japan 3 Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan 4 Heartland Assays Inc., Ames, IA, USA 5 Boston University School of Medicine, Boston, Massachusetts, USA 6 Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, Nanko-naka, Suminoe-ku, Osaka, Japan Introduction 1a,25-Dihydroxyvitamin D 3 (1a,25(OH) 2 D 3 ), the active form of vitamin D 3 , mediates its biological effects by binding to the vitamin D receptor (VDR) [1–4]. Activation of the VDR leads to the expression of genes involved in bone and calcium metabolism, cellular prolif- eration and differentiation, immune responses, etc. [5]. Thus, 1a,25(OH) 2 D 3 and its analogs have been developed for the clinical treatment of rickets, osteoporosis, Keywords crystal structure; cytochrome P450; electron transport chain; protein engineering; vitamin D Correspondence T. Sakaki, Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Kurokawa, Imizu, Toyama, Japan Fax: +81 766 56 2498 Tel: +81 766 56 7500 E-mail: tsakaki@pu-toyama.ac.jp Database Structural data are available in the Protein Data Bank database under the accession numbers 3CV8 and 3CV9 (Received 14 May 2010, revised 1 July 2010, accepted 27 July 2010) doi:10.1111/j.1742-4658.2010.07791.x Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V ⁄ R84A, has a high ability to convert vitamin D 3 to its biologically active form, 1a,25-dihydroxyvitamin D 3 [1a,25(OH) 2 D 3 ], suggesting the possibility for R73V ⁄ R84A to produce 1a,25(OH) 2 D 3 . Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V ⁄ R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25-hydroxyvitamin D 3 [25(OH)D 3 ] and 1a,25(OH) 2 D 3 were detected in the cell culture of the recombinant S. lividans. A large amount of 1a,25(OH) 2 D 3 , the second-step metabolite of vitamin D 3 , was observed, although a considerable amount of vitamin D 3 still remained in the culture. In addition, novel polar metabolites 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 , both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5 : 1. The crystal structure of the double variant with 1a,25(OH) 2 D 3 and a docking model of 1a,25(OH) 2 D 3 in its active site strongly suggest a hydrogen-bond network including the 1a-hydroxyl group, and several water molecules play an important role in the substrate- binding for 26-hydroxylation. In conclusion, we have demonstrated that R73V ⁄ R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D 3 to produce biologically useful compounds. Abbreviations CYP, cytochrome P450; 25(OH)D 3, 25-hydroxyvitamin D 3 ;1a(OH)D 3 ,1a-hydroxyvitamin D 3 ;1a,25(OH) 2 D 3 ,1a,25-dihydroxyvitamin D 3 ; 1a,25,26(OH) 3 D 3 ,1a,25,26-trihydroxyvitamin D 3 ; FDR, ferredoxin reductase; FDX, ferredoxin. FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS 3999 psoriasis, secondary hyperparathyroidism, autoimmune diseases and cancers [6,7]. The large-scale production of 1a,25(OH) 2 D 3 from vitamin D 3 uses a bioconversion system of Amycola- ta autotrophica, which is one of the successful applica- tions of the P450 enzymatic reaction, on an industrial scale [8]. The primary structure of the cloned gene reveals that the 25-hydroxylase of vitamin D 3 is a water-soluble cytochrome P450 named CYP105A2 [9]. Recently, Fujii et al. [10] demonstrated that Pseudono- cardia autotrophica P450, a member of the CYP107 family, could convert vitamin D 3 to 1a,25(OH) 2 D 3 . We found that Streptomyces griseolus CYP105A1, which has 55% amino acid homology to CYP105A2, also has weak activities of both 25-hydroxylation and 1a-hydroxylation of vitamin D 3 to produce 1a,25(OH) 2 D 3 [11]. Recent crystal-structure analysis of CYP105A1 revealed three arginine residues (Arg73, Arg84 and Arg193) within the substrate-binding pocket of CYP105A1 [12]. The Ala-scan mutation analysis indicated that the variant with R73A and R84A showed much higher activity than the wild type, suggesting that Arg73 and Arg84 residues may have an inhibitory effect on activity, while Arg193 may be essential for activity. We therefore attempted to further enhance the vitamin D hydroxylation activity of CYP105A1 by mutating these two inhibitory Arg resi- dues into various amino acids. The resulting double- variant R73V ⁄ R84A exhibited 435- and 110-fold higher k cat ⁄ K m values, respectively, for 1a-hydroxyvita- min D 3 [1a(OH)D 3 ] 25-hydroxylation and 25-hydrox- yvitamin D 3 [25(OH)D 3 ]1a-hydroxylation, compared with the wild-type enzyme [13]. These values notably exceed those of CYP27A1, which is a physiologically essential vitamin D 3 hydroxylase. The results suggest that the R73V ⁄ R84A variant could be useful for the bioconversion process to produce 1a,25(OH) 2 D 3 from vitamin D 3 . In this study, we expressed the R73V ⁄ R84A variant in Streptomyces lividans cells, and used the recombi- nant cells for the bioconversion of vitamin D 3 to its hydroxylated metabolites. As expected, the cells pro- duced 1a,25(OH) 2 D 3 . Furthermore, a polar peak was also detected. In this article we describe the identifica- tion of the novel metabolites, and the mechanism of a three-step hydroxylation by the R73V ⁄ R84A variant. In addition, we evaluated the biological significance of the recombinant S. lividans cells expressing the R73V ⁄ R84A variant from the viewpoint of industrial and medical applications. Results Expression of the R73V ⁄ R84A variant of CYP105A1 in the recombinant S. lividans TK23 cells The R73V ⁄ R84A variant of CYP105A1 was expressed in the recombinant S. lividans cells (Fig. 1). The expression level of R73V ⁄ R84A, 72 h after addition of the substrate, was estimated to be approximately 3 lmolÆL )1 of culture based on western blot analysis using R73V ⁄ R84A purified from recombinant Escheri- chia coli cells as a standard (Fig. 2). Metabolism of vitamin D 3 in the recombinant S. lividans cell culture Figure 3 shows HPLC profiles of vitamin D 3 and its metabolites. Their retention times and mass spectra (data not shown) strongly suggest that the two major metabolites are 25(OH)D 3 and 1a,25(OH) 2 D 3 ,. These metabolites were not observed in the control S. livi- dans cells, suggesting that they were produced by the R73V ⁄ R84A variant expressed in the recombinant S. lividans TK23 cells. These results are consistent with the results of our previous studies showing that the R73V ⁄ R84A variant has the capability to convert vitamin D 3 into 1a,25(OH) 2 D 3 through 25(OH)D 3 . However, a novel metabolite, which is more polar than 1a,25(OH) 2 D 3 , was observed in this study. The conversion ratios of 25(OH)D 3 ,1a,25(OH) 2 D 3 and the more polar metabolite at 24 h were 38.9, 10.6 and 2.7%, respectively, as shown in Fig. 3. The amount of 25(OH)D 3 showed a maximum at 24h, and then decreased, while the amount of 1a,25(OH) 2 D 3 gradually increased. The conversion ratios of Fig. 1. Structure of the expression plasmid for the CYP105A1 vari- ant (R73V ⁄ R84A), FDX1 and FDR1. The DNA fragment harboring three genes was inserted into HindIII and EcoRI sites of the vector pIJ6021, as described in the Materials and methods. Multi-hydroxylations of vitamin D 3 K. Hayashi et al. 4000 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS 1a,25(OH) 2 D 3 at 48 and 72 h were 11.8% and 15.2%, respectively (Fig. 4). The most polar metabolite increased linearly, and its conversion ratios at 48, 72 and 96 h were 4.7%, 8.2% and 10%, respectively. Judging from the time course of these metabolites, the most polar metabolite appears to be a final product derived from vitamin D 3 via 25(OH)D 3 and 1a,25(OH) 2 D 3 . Identification of the most polar metabolite As shown in Fig. 5, a molecular ion of m ⁄ z 433 (M+H) is very small compared with major frag- ment ions of m ⁄ z 415 (M+H-H 2 O) and m⁄ z 397 (M+H-2H 2 O), suggesting that this metabolite has a 1a-hydroxyl group. Based on these results, it appears that this metabolite occurs through the further hydrox- ylation of 1a,25(OH) 2 D 3 . 1 NMR studies revealed that a signal at d1.2 ppm (6H, singlet) derived from 26,27- CH 3 observed in 1a,25(OH) 2 D 3 was not observed in the metabolite M3, suggesting that C26 or C27 of 1a,25(OH) 2 D 3 was hydroxylated to yield M3 (data not shown). These results led us to speculate that M3 could be 1a,25(R),26(OH) 3 D 3 or 1a,25(S),26(OH) 3 D 3 . To confirm this, we examined periodate oxidation that would cleave the C25–C26 bond of M3 to yield 25-oxo-27-nor-1a (OH)D 3 . After the periodate treat- ment of M3, the product was eluted at 24.1 min, while M3 was eluted at 18.9 min under the HPLC conditions described in the Materials and methods. The product recovered from the HPLC eluents was analyzed using LC-MS. As shown in Fig. 5, its mass spectrum showed a molecular ion of m ⁄ z 401 (M+H) and fragment ions of m ⁄ z 383 (M+H-H 2 O) and m ⁄ z 365 (M+H-2H 2 O), indicating that this compound is 25-oxo-27-nor-1a Fig. 3. HPLC profiles of vitamin D 3 and its metabolites formed in the recombinant Streptomyces lividans TK23 cells. After culture for 48 h with 20 mgÆL )1 (0.05 mM) of vitamin D 3 and 0.2% 2-hydroxy- propyl-b-cyclodextrin, the cell suspension was extracted and analyzed by HPLC, as described in the Materials and methods. Fig. 2. Western blot analysis of R73V ⁄ R84A expressed in S. livi- dans TK23 cells. After 72 h of culture, cell lysate prepared from 1 lL of whole-cell culture was separated by SDS ⁄ PAGE and reacted with an antiserum against CYP105A1 after electrophoretic transfer to a nitrocellulose filter. Lane 1, purified sample of R73V ⁄ R84A (1 pmol) prepared from the recombinant Escherichia coli cells; lane 2, the above-mentioned cell lysate. The numbers indicate the migration points of the prestained protein marker pro- teins (Cell Signaling Technology Inc., MA, USA): fusion of maltose- binding protein (MBP) and paramyosin, M r 80,000; fusion of MBP and chitin-binding protein (CBD), M r 58,000; rabbit muscle aldolase, M r 46,000; E. coli triosephosphate isomerase, M r 30,000; and CBD-BmFKBP13, M r 25,000. Fig. 4. Time courses of the vitamin D 3 metabolites, 25(OH)D 3 ( ), 1a,25(OH) 2 D 3 (d), and the metabolite M3 ( ) in the recombinant Streptomyces lividans TK23 cells expressing R73V ⁄ R84A. K. Hayashi et al. Multi-hydroxylations of vitamin D 3 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS 4001 (OH)D 3 , derived from 1a,25(R), 26(OH) 3 D 3 or 1a,25(S),26(OH) 3 D 3 . Finally, we performed co-chroma- tography of M3 with authentic standards of 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 using a chiral column. As shown in Fig. 6, the retention time of M3 coincides with that of 1a,25(R),26(OH) 3 D 3 , and co-chromatography of M3 with authentic stan- dards of 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 confirmed that the dominant component of M3 is 1a,25(R),26(OH) 3 D 3 . However, the peak of M3 shows a shoulder, probably because of the presence of 1a,25(S),26(OH) 3 D 3 . The ratio of 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 was estimated to be 5 : 1, based on HPLC data containing authentic standards of 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 at various ratios (Fig. 6F). Fig. 5. Mass spectra of the metabolite M3 (A) and its periodate-oxidation product (B). The putative structure of the periodate-oxi- dation product is shown. Fig. 6. HPLC analysis of M3 using a chiral b-cyclodextrin column. The metabolite M3, authentic standards of 1a,25(R),26(OH) 3 D 3 (R-form), and 1a,25(S),26(OH) 3 D 3 (S-form) were analyzed alone or in combination, as follows: R-form (400 pmol) (A); S-form (400 pmol) (B); R-form (200 pmol) and S-form (200 pmol) (C); R-form (200 pmol) and S-form (200 pmol) + M3 (80 pmol) (D); M3 (150 pmol) (E); and R-form (200 pmol) (A) and S-form (40 pmol) (F). Multi-hydroxylations of vitamin D 3 K. Hayashi et al. 4002 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS Metabolism of 1a,25(OH) 2 D 3 by the R73V ⁄ R84A variant expressed in E. coli cells To confirm the 26-hydroxylation activity of the R73V ⁄ R84A variant, the in vitro reaction, including the R73V ⁄ R84A variant and 1a,25(OH) 2 D 3 , was examined. In this experiment, overexpressed R73V ⁄ R84A variant in E. coli and the reconstitution system was used. The reaction mixture was extracted, and then analyzed by HPLC using both an ODS column and the chiral column. As expected, the metab- olite showed the same retention time as the metabolite M3 prepared from the recombinant S. lividans TK23 cell suspension, suggesting that the R73V ⁄ R84A variant catalyzes hydroxylation of 1a,25(OH) 2 D 3 (Fig. 7). The HPLC analysis using the chiral column showed nearly the same peak with a shoulder, as shown in Fig. 7B. These results strongly suggest that the R73V ⁄ R84A variant has the capability to convert 1a,25(OH) 2 D 3 to 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 at a ratio of 5 : 1. Comparison of kinetic parameters of the R73V ⁄ R84A variant-dependent 25-, 1a and 26-hydroxylation activities towards vitamin D 3 , 25(OH)D 3 ,1a(OH)D 3 and 1a,25(OH) 2 D 3 The K m and k cat of the R73V ⁄ R84A variant for vita- min D 3 25-hydroxylation were estimated to be 3.5 lm and 0.141 min )1 , respectively (Table 1). A small amount of 1a(OH)D 3 was detected, demonstrating that the R73V ⁄ R84A variant has vitamin D 3 1a-hydroxyl- ation activity. However, the kinetic parameters were not estimated correctly, because the resultant 1a(OH)D 3 is readily converted to 1a,25(OH) 2 D 3 ,as suggested by a much higher k cat ⁄ K m value for 1a(OH)D 3 25-hydroxylation than for any other reac- tions detailed in Table 1. The K m and k cat of the R73V ⁄ R84A variant for 25(OH)D 3 1a-hydroxylation were estimated to be 2.2 lm and 0.136 min )1 , respec- tively [12] (Table 1), while 25(OH)D 3 26-hydroxylation activity was not observed. Regarding the metabolism of 1 a(OH)D 3 , the R73V ⁄ R84A variant prefers 25-hydroxylation to 26-hydroxylation, judging from no detection of 1a,26(OH) 2 D 3 (Table 1). Therefore, among the four substrates examined in this study, 26-hydroxylation was only observed in the metabolism of 1a,25(OH) 2 D 3 . The kinetic parameters K m and k cat of the R73V ⁄ R84A variant for hydroxylation at C26 or C27 of 1a,25(OH) 2 D 3 to yield 1a,25,26(R)(OH) 3 D 3 and 1a,25,26(S)(OH) 3 D 3 were estimated to be 2.2 lm and 0.136 min )1 , respectively (Table 1). It should be noted that the k cat ⁄ K m value for 1a,25(OH) 2 D 3 26-hydroxylation is similar to those for vitamin D 3 25-hydroxylation and for 25(OH)D 3 1a-hydroxylation. Comparison of the stability of the oxygenated form of wild type and the double variants of CYP105A1 Our previous studies revealed that the double variants of CYP105A1 – R73A ⁄ R84A and R73V ⁄ R84A – have much higher activity than the wild-type CYP105A1. One of the most essential reasons for the high activity appears to be a significant increase in the coupling effi- ciency between product formation and NADPH oxida- tion. Therefore, we examined the stability of the oxygenated forms of the wild-type CYP105A1 and its variants. Figure 8 shows spectral analysis of the NADPH- dependent reduction of heme iron of the P450s. Although the wild-type CYP105A1 showed a rapid conversion of P450 to P420, the double variants R73A ⁄ R84A and R73V ⁄ R84A showed a peak at 450 nm, suggesting that the oxygenated forms of these Table 1. Kinetic parameters of the R73V ⁄ R84A variant of CYP105A1. VD 3 , vitamin D 3. ND, kinetic parameters could not be determined although the activity was detected; –, the activity was not detected. Substrate Hydroxylation K m (lM) k cat (min )1 ) k cat ⁄ K m VD 3 25 3.5 0.141 0.040 VD 3 1a ND ND ND VD 3 26 – – – 25(OH)D 3 1a 2.2 0.136 0.062 25(OH)D 3 26 – – – 1a(OH)D 3 25 6.5 2.12 0.33 1a(OH)D 3 26 – – – 1a,25(OH) 2 D 3 26 1.2 0.051 0.043 Fig. 7. HPLC analysis of the metabolite M3 produced by R73V ⁄ R84A expressed in Escherichia coli cells. 1a,25(OH) 2 D 3 and its metabolite, M3, were analyzed by RP-HPLC (A), and the chiral b-cyclodextrin column (B) as described in the Materials and methods. K. Hayashi et al. Multi-hydroxylations of vitamin D 3 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS 4003 variants are more stable than the wild type. This sta- bilization was also observed in the single variant R84A, whereas R73A showed no such stabilization. Thus, the stabilization of the oxygenated form origi- nates from the mutation of Arg84. Discussion Our new discoveries in this study are summarized as follows. First, the double variants of CYP105A1 have 26-hydroxylation activity towards 1a,25(OH) 2 D 3 . Thus, they catalyze a three-step hydroxylation at C25, C1 and C26 (C27) positions of vitamin D 3 to yield 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 . Second, the putative reasons why the double variants have much higher activities than the wild type were demon- strated. (a) The mutation of Arg84 to alanine stabilizes the oxygenated form of P450, thus increasing the cou- pling efficiency between product formation and NADPH oxidation and (b) the mutation of Arg73 to alanine or valine produces a hydrogen-bond network formed by the re-arrangement of water molecules sur- rounding the 1a-hydroxyl group. Third, the recombi- nant S. lividans cells expressing the R73V ⁄ R84A variant of CYP105A1 are promising candidates for producing the active forms of vitamin D 3 as useful drugs. In this study, we selected the R73V ⁄ R84A variant as a catalyst and S. lividans as a host for the following reasons (a) S. lividans has been utilized as a potential host for protein production, (b) the codon usage and GC content of the CYP105A1 gene are similar to those of S. lividans genomic DNA, (c) any ferredoxins of S. lividans and their reductases may act as an elec- tron donor for the R73V ⁄ R84A variant based on the fact that CYP105A2 showed vitamin D 3 25-hydroxyl- ation activity in S. lividans cells [9] and (d) Actinomy- cetes, including Streptomyces, exhibit potential advantages in the synthesis of secondary metabolites of industrial and medical importance in the bioconver- sion processes. Consequently, S. lividans TK23 cells and the vector pIJ6021 were used as a host–vector system, and the R73V ⁄ R84A variant was expressed under the control of the tipA promoter induced by thiostrepton. The metabolites of vitamin D 3 were detected in the recombinant S. lividans cell culture. Unexpectedly, a large amount of 1a,25(OH) 2 D 3 , the second-step metabolite of vitamin D 3 , was observed, although most of the added vitamin D 3 still remained in the culture. In addition, a novel metabolite peak was observed (Fig. 3). These results appear to be inconsistent with our previous in vitro studies using recombinant enzymes in a reconstituted system [13]. However, it is possible that the efficiency of cell uptake is different among vitamin D 3 , 25(OH)D 3 and 1a,25(OH) 2 D 3 , based on their hydrophobicity and affinity for 2-hydroxypropyl-b-cyclodextrin added to the culture. Among the three compounds, the uptake of vitamin D 3 into S. lividans cells appears to be the least efficient. By contrast, 1a,25(OH) 2 D 3 appears to read- ily cross the cell membrane and enter the cell. In addition to 25(OH)D 3 and 1a,25(OH) 2 D 3 , we found novel metabolites – 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 – which were identified by LC-MS analysis with periodate treatment, NMR studies and HPLC analysis using a chiral column. It should be noted that 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 can be separated by using the chiral column, although the previous separation method required acetylation [14]. R73V ⁄ R84A and R73A ⁄ R84A prepared from the recombinant E. coli cells also showed the conversion of 1a,25(OH) 2 D 3 to Fig. 8. NADPH-dependent reduced CO-difference spectra of CP105A1 (A) and the R73A ⁄ R84A variant (B). NADPH-dependent reduced CO-difference spectra were measured at 15-second inter- vals in the presence of 2 l M wild-type CP105A1 (A) or the R73A ⁄ R84A variant (B), 0.02 mgÆmL )1 of spinach ferredoxin, 0.02 UÆmL )1 of spinach ferredoxin-reductase, 10 l M of 1a(OH)D 3 , and 1 m M NADPH. Very rapid conversion from P450 to P420 was observed in the wild-type CP105A1. By contrast, R73A ⁄ R84A showed a P450 peak, indicating that the oxygenated form of R73A ⁄ R84A is more stable than the wild type. Multi-hydroxylations of vitamin D 3 K. Hayashi et al. 4004 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 . These results confirm that these variants have 26-hydroxyl- ation activity towards 1a,25(OH) 2 D 3 . In our previous studies, 1a,25(OH) 2 D 3 was considered to be the final product, but 1a,25(OH) 2 D 3 was found to be a sub- strate for 26-hydroxylation in this study. Figure 9 shows the reported crystal structure with 1a,25(OH) 2 D 3 [Protein Data Bank (PDB) code 3cv9] [13] which is superimposed by a docking model of 1a,25(OH) 2 D 3 in the active site for 26-hydroxylation. The distance (13.5 A ˚ ) from C26 to heme iron in the crystal structure appears to be too far to simulate the reactive structure of the enzyme–substrate for 26- hydroxylation. Thus, we need to construct a docking model for the 26-hydroxylation. As shown in Fig. 9, the distance (3.6 A ˚ ) from C26 to heme iron in the docking model is suitable for the 26-hydroxylation. A small rotation of a secosteroid skeleton, and a considerable conformational change of the side chain in the heme pocket, will make the 26-hydroxylation possible. It is noted that Fig. 9 shows a docking model to yield more 1a,25(R),26(OH) 3 D 3 than 1a,25(S),26(OH) 3 D 3 . It is possible that the distance from C26 or C27 to heme iron, and the orientations of the hydrogen atoms at C26 or C27, decide the ratio between 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 . Regarding the metabolism of 25(OH)D 3 , we did not detect any 25,26(OH) 2 D 3 , and only 1a,25(OH) 2 D 3 was observed as a metabolite. This strongly suggests that the 1a-hydroxyl group plays an important role in the 26-hydroxylation. As shown in Fig. 6, the 1a-hydroxyl group forms a hydrogen bond with a water molecule with the distance of 2.9 A ˚ . In addition, the bound water molecule forms a hydrogen-bond network involving the A92 amide group, the Q93 side chain and four other waters in the heme pocket. Therefore, it is possible that this hydrogen-bond network, includ- ing the 1a-hydroxyl group, is responsible for the stable binding of 1a,25(OH) 2 D 3 for 26-hydroxylation. In a previous report, we proposed that the reason for increased activity by the mutation on R73 might be the direct effect of its size and charge on the confor- mation of the hydrophobic substrate in the large active-site pocket. Because the single variant R84A (PDB code 2zbz, 1.9A ˚ resolution) does not have this hydrogen-bond network [12], it implies that the hydro- gen-bond network formed by the re-arrangement of the water molecule surrounding the 1a-hydroxyl group is associated with the high activity of R73 variants. Figure 10 summarizes the metabolic pathways of vitamin D 3 by the R73V ⁄ R84A variant. Although the first step contains both 25-hydroxylation and 1a-hydroxylation, R73V ⁄ R84A prefers the former. The minor product, 1a(OH)D 3 , is a good substrate of R73V ⁄ R84A for the 25-hydroxylation to yield 1a,25(OH) 2 D 3 . It seems likely that the 1a-hydroxyl group of 1a(OH)D 3 forms a hydrogen bond with the bound water molecule as well as 1a-hydroxyl group of Fig. 9. Hydrogen-bond network involving the 1aOH group of 1a,25(OH) 2 D 3 and water molecules observed in the crystal struc- ture of the R73A ⁄ R84A variant. The docking model of 1a,25(OH) 2 D 3 for 26-hydroxylation is superposed and shown in green. The water molecules are shown as red spheres. The hydro- gen bonds are shown as broken lines. Fig. 10. Metabolic pathways of vitamin D 3 catalyzed by the R73V ⁄ R84A variant of CYP105A1. K. Hayashi et al. Multi-hydroxylations of vitamin D 3 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS 4005 1a,25(OH) 2 D 3 (Fig. 9). However, it should be noted that R73V ⁄ R84A much prefers 25-hydroxylation to 26-hydroxylation towards 1a(OH)D 3 . Accordingly, both pathways of the second steps produce 1a,25(OH) 2 D 3 . In this study, we found the third step, which con- verts 1a,25(OH) 2 D 3 into 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 . Thus, the major pathway occurs in the order of 25-hydroxylation, 1a-hydroxylation and 26-hydroxylation. It is possible to assume that the first product, 25(OH)D 3 , is released from the heme pocket and re-enters the heme pocket in the opposite direction for 1a-hydroxylation, and the resultant product, 1a,25(OH) 2 D 3 , is released from the heme pocket and again re-enters the heme pocket in the opposite direc- tion for 26-hydroxylation. It is well known that 1a,25(OH) 2 D 3 has potent anti- proliferative effects in many cancer-cell types, includ- ing breast and prostate cancers. However, 1a,25(OH) 2 D 3 is not suitable as a therapeutic agent for cancer treatment, because its systemic administration causes hypercalcemia and hypercalciuria. Therefore, a less calcemic vitamin D analog is promising for cancer treatment. As reported previously, the calcemic effect of 1a,25(R),26(OH) 3 D 3 or 1a,25,26-trihydroxyvitamin D 3 [1a,25,26(OH) 3 D 3 ] is significantly lower than that of 1a,25(OH) 2 D 3 , whereas its antiproliferative activity is nearly the same [15–18]. These findings suggest that 1a,25(R),26(OH) 3 D 3 is suitable for anticancer treat- ment. Although conversion of 1a,25(OH) 2 D 3 into 1a,25(R),26(OH) 3 D 3 and 1a,25(S),26(OH) 3 D 3 is unfa- vorable for practical use of the recombinant S. lividans cells to produce 1a,25(OH) 2 D 3 , it is quite favorable for the production of a promising anti-cancer compound. Recently, Peng et al. [19] and Lou et al. [20] claimed that 25(OH)D 3 was able to act as a hormone in mam- mary gland using CYP27B1 [25(OH)D 3 1a-hydroxyk- ase] knockout mice. They concluded that 25(OH)D 3 could be developed as a nontoxic, natural, chemopre- ventive agent for further development for cancer pre- vention. Based on these results, all three compounds produced in this study appear to be clinically useful. In this study, we attempted and succeeded in estab- lishing a bioconversion system to convert vitamin D 3 into its multiple hydroxylated metabolites, including 25(OH)D 3 ,1a,25(OH) 2 D 3 and 1a,25,26(OH) 3 D 3 ,by using recombinant S. lividans cells expressing a variant of CYP105A1. It should be noted that we did not con- firm the expression of ferredoxin 1 (FDX1) and ferre- doxin reductase 1 (FDR1) genes on the expression plasmid. Recombinant S. lividans cells harboring a plasmid containing the R73V ⁄ R84A variant of CYP105A1, and FDX1 genes without the FDR1 gene, showed activity that was not so different from those harboring a plasmid containing three genes. These results strongly suggest that some endogenous FDR(s) of S. lividans function as electron donors. In order to enhance the productivity of these metab- olites, we plan to further mutate CYP105A1, increase its expression level, optimize the electron-transfer sys- tem and modify culture conditions. Materials and methods Materials DNA-modifying enzymes, restriction enzymes and E. coli SCS110 were purchased from Takara Shuzo Co. Ltd (Kyoto, Japan). S. lividans TK23 cells and the vector pIJ6021 [21] were kindly provided by Dr Hiroyasu Onaka of Toyama Prefectural University. The HPLC column SU- MICHIRAL OA-7000 (4.6 · 250 mm) was purchased from Sumika Chemikcal Analysis Service, Ltd (Osaka, Japan). Ferredoxin and NADPH-ferredoxin reductase from spinach were purchased from Sigma (St Louis, MO, USA). Vitamin D 3 ,1a(OH)D 3 ,1a,25(OH) 2 D 3 , glucose dehydrogenase, catalase, NZ-casein, NZ-amine, corn steep liquor and 2-hydroxypropyl-b-cyclodextrin were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). 25(OH)D 3 was purchased from Funakoshi Co. Ltd (Tokyo, Japan). 1a,25(R),26(OH) 3 D 3 and 1 a,25(S),26(OH) 3 D 3 were chemi- cally synthesized, as described by Partridge et al. [14]. Meat extract was purchased from Kyokuto Pharmaceuticals Co. (Tokyo, Japan). Bacto-soytone, bacto-peptone and yeast extract were obtained from Difco Laboratories (Detroit, MI, USA). S. griseolus CYP105A1 and ferredoxin genes were kindly provided by Sumitomo Chemical Co. Ltd (Takarazuka, Japan). NADPH was purchased from Orien- tal Yeast Co. Ltd (Tokyo, Japan). Other chemicals used were of the highest quality commercially available. Expression of the R73V ⁄ R84A variant of CYP105A1 in S. lividans TK23 cells Variants were generated using the Quick ChangeÔ Site- directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) using the CYP105A1 gene as a template. The oligonucleo- tide primers used for mutagenesis to yield the variant R73V ⁄ R8A were as described previously [13]. For construc- tion of the expression plasmid, E. coli SCS110, which has no ability to methylate DNA, was used as a host. The PCR fragment containing R73V ⁄ R84A and FDX1 genes, with HindIII and PstI restriction sites, was prepared using the primers 5¢-ACCAAGCTTATGAAAAGATACCGCCAC- GACG-3¢ and 5 ¢-TTCTGCAGTCACCAGGTGACCGG- GAGTTCG- 3¢, and the expression plasmid for R73V ⁄ R84A in E. coli cells as a template DNA. The resul- tant PCR fragment was digested with HindIII and PstI, Multi-hydroxylations of vitamin D 3 K. Hayashi et al. 4006 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS and inserted into the HindIII and PstI sites of pUC19. The PCR fragment containing the Streptomyces coelicolor FDR1 gene [22], with PstI and EcoRI restriction sites, which contains approximately 170 bp 5¢ upstream region from the initial codon ATG, was prepared using the prim- ers 5¢-AACTGCAGCCGTCCCCACGCCTGCGTCACC-3¢ and 5¢-CGAATTCTCAGGCGCCGCTCTCGCGGAGCA- 3¢, and total DNA of S. coelicolor cells as a template. The resultant PCR fragment was digested with PstI and EcoRI, and inserted into the PstI and EcoRI sites of pUC19. Then, the HindIII ⁄ PstI fragment containing R73V ⁄ R84A and FDX1 genes, and the PstI ⁄ EcoRI fragment containing the FDR1 gene prepared from the cloning vectors was dou- bly inserted into HindIII and EcoRI sites of the vector pIJ6021. The resultant co-expression plasmid for R73V ⁄ R84A, FDX1 and FDR1 was introduced into protoplasts of S. liv- idans TK23 according to the method described by Horinou- chi et al. [23]. After 3 days of incubation at 30 °C, kanamycin-resistant colonies were obtained. Culture of the recombinant S. lividans cells For the preculture, the transformant on Bennett’s agar plate containing 0.1% yeast extract, 0.1% meat extract, 0.2% NZ-amine, 1% maltose, 2% agar and kanamycin at a final concentration of 15 lgÆmL )1 was inoculated into medium (10 mL) containing 1% soluble starch, 0.5% glucose, 0.3% NZ-casein, 0.2% yeast extract, 0.5% Bacto- peptone, 0.1% KH 2 PO 4 , 0.05% MgSO 4 Æ7H 2 O, 0.3% CaCO 3 and kanamycin at a final concentration of 15lg ⁄ ml, and then vigorously shaken at 30 °C for 3 days. The preculture (2ml) was inoculated into the sterilized medium (100 mL) containing 1.5% glucose, 1.5% Difco bacto Soytone, 0.5% Corn steep liquor, 0.5% NaCl, 0.2% CaCO 3 and kanamycin at a final concentration of 15lg ⁄ ml, and incubated on a rotary shaker for 3 days at 200 rpm and 30 °C. Thiostrepton was then added into the medium at a final concentration of 10 l g ⁄ ml to induce expression of R73V ⁄ R84A variant. HPLC analysis of vitamin D 3 metabolites produced in the recombinant S. lividans cells Twenty-four hours after the addition of thiostrepton, 2-hy- droxypropyl-b-cyclodextrin solution (200 mgÆmL )1 of water) and vitamin D 3 -ethanol solution (20 mg in 0.5 mL of etha- nol) were added to 100 mL of culture of the recombinant S. lividans cells. Aliquots of the culture were extracted at 0, 24, 48 and 72 h with four volumes of c hloroform ⁄ methanol (3 : 1, v ⁄ v). The organic phase was recovered and dried in a vacuum evaporator centrifuge (Sakuma Seisakusyo, Tokyo, Japan). The resulting residue was solubilized with acetonitrile and applied to HPLC under the following con- ditions: column, YMC-Pack ODS-AM (4.6 · 300 mm) (YMC Co., Tokyo, Japan); UV detection, 265 nm; flow rate, 1.0 mLÆmin )1 ; column temperature, 40 °C; mobile phase, a linear gradient of 70-100% acetonitrile aqueous solution in 15 min followed by 100% acetonitrile for 25 min. The metabolite designated as M3, which was recovered from the RP-HPLC, was further analyzed by HPLC using a chiral b-cyclodextrin column under the following condi- tions: column, SUMICHIRAL OA-7000 (4.6 · 250 mm) (Sumika Chemikcal Analysis Service, Ltd); UV detection, 265 nm; flow rate, 0.7 mLÆmin )1 ; column temperature, 25 °C; mobile phase, methanol ⁄ water (85 : 15, v ⁄ v). LC-MS analysis of metabolites produced in the recombinant S. lividans cells Isolated metabolites from HPLC effluents were subjected to mass spectrometric analysis, using a Finnigan LCQ ADVANTAGE MIX (ThermoFisher SCIENTIFIC, Wal- tham, MA, USA) with atmospheric pressure chemical ioni- zation in the positive mode. The conditions of LC were: column, reverse-phase ODS column (2 · 150 mm, Develosil ODS-HG-3; Nomura Chemical Co. Ltd, Aichi, Japan); mobile phase, acetonitrile ⁄ methanol ⁄ water (3 : 4 : 3, v ⁄ v ⁄ v); flow rate, 0.2 mLÆmin )1 ; UV detection, 265 nm. Periodate oxidation Periodate oxidation was carried out to identify the presence of vicinal diol or an a-keto group. The metabolite M3 was dissolved in 15 lL of methanol, and 10 lL of 5% sodium metaperiodate aqueous solution was added as described by Reinhardt et al. [24]. After 30 min of incubation at 25 °C, the solution was dried in vacuo and the resultant residue was analyzed by HPLC and LC-MS. The HPLC conditions were as follows: column, YMC-Pack ODS-AM (4.6 · 300 mm) (YMC Co.); UV detection, 265 nm; flow rate, 1.0 mLÆmin )1 ; column temperature, 40 °C; mobile phase, a linear gradient of 20–100% acetonitrile aqueous solution per 25 min fol- lowed by 100% acetonitrile for 12 min. 1 H-NMR analysis of the metabolite produced by the recombinant S. lividans cells The 400-MHz 1 H-NMR spectra of the metabolite M3 was measured on a BRUKER-400 ( 1 H, 399.9 MHz). The metabolite M3 was dissolved in 500 lLofCD 3 OD and transferred into a probe. Preparation of anti-CYP105A1 serum Purified wild-type CYP105A1 (0.5 mg) was mixed with an equal volume of Freund’s complete adjuvant, and then injected subcutaneously into a young male rabbit weighing K. Hayashi et al. Multi-hydroxylations of vitamin D 3 FEBS Journal 277 (2010) 3999–4009 ª 2010 The Authors Journal compilation ª 2010 FEBS 4007 2.5 kg. After 2 and 4 weeks, the rabbit was boosted with 1.0 mg of the antigen in emulsified Freund’s incomplete adjuvant, and bled 1 week after the final injection. Western blot analysis of the R73V ⁄ R84A variant of CYP105A1 expressed in S. lividans cells Cytosolic fractions prepared from recombinant S. lividans cells were subjected to SDS ⁄ PAGE on a 10% gel and trans- ferred to a poly(vinylidene difluoride) membrane. Immun- odetection was performed using the anti-CYP105A1 serum as the primary antibody, and alkaline phosphatase-conju- gated goat anti-rabbit IgG as the secondary antibody. Spectral analysis The reduced CO-difference spectra of the R73V ⁄ R84A vari- ant expressed in E. coli cells were measured using a Hitachi U-3310 spectrophotometer with a head-on photomultiplier (Tokyo, Japan) [25]. The P450 content of the purified wild- type or variants of CYP105A1 was estimated using a molar extinction coefficient of 110 mm )1 Æcm )1 at 417 nm [11]. To measure the NADPH-dependent reduction rate of heme iron, reduced CO-difference spectra were measured at 15-second intervals in the presence of 2 lm wild-type or variants of CYP105A1, 0.02 mgÆmL )1 of FDX, 0.02 UÆmL )1 of FDR, 10 lm 1a(OH)D 3 and 1 mm NADPH. Metabolism of 1a,25(OH) 2 D 3 by the R73V ⁄ R84A variant expressed in E. coli cells The metabolism of 1a,25(OH) 2 D 3 was examined in a recon- stituted system containing 0.2 lm R73V ⁄ R84A, 0.1 mgÆmL )1 of spinach ferredoxin, 0.1 UÆmL )1 of spinach ferredoxin reductase, 1 UÆmL )1 of glucose dehydrogenase, 1% glucose, 0.1 mgÆmL )1 of catalase, 1 mm NADPH, 0.5–10.0 lm of the substrate, 100 mm Tris ⁄ HCl (pH7.4) and 1 mm EDTA, at 30 °C, as described previously [13]. Other methods The concentrations of vitamin D 3 derivatives were esti- mated by their molar extinction coefficient of 1.80 · 10 4 m )1 Æcm )1 at 264 nm [26]. Docking of 1a,25(OH) 2 D 3 to the variant enzyme was performed by procedures described previously [12] using the Lamarckian Genetic Algorithm (LGA) method implemented in Autodock 4 [27]. The model of the R73V ⁄ R84A variant was generated from the crystal structure of R73A ⁄ R84A (PDB code 3cv9), and used as a template for the docking calculations. The hydrogenated protein and atomic charges of the residues were calculated using the method described in the Autodock tools package. The 1a,25(OH) 2 D 3 was treated as a flexible ligand, and the side chains of V73, F85, V88 and Q93 were treated as a flexible residue. Water molecules numbered Wat560, 587 and 593 in the binding site were included in the calculation. Cluster analysis was performed on the docked results of 100 runs using a root-mean-square tolerance of 2.0 A ˚ . Other parameters were left at the default values. The figure of protein structure was prepared using pymol software (http://www.pymol.org). Acknowledgement This work was supported, in part, by a Ministry of Education, Culture, Sports, Science and Technology grant, and the Novozymes Japan Research Fund. References 1 Norman AW, Myrtle JF, Midgett RJ, Nowicki HG, Williams V & Popjak G (1971) 1,25-dihydroxycholecal- ciferol: identification of the proposed active form of vitamin D3 in the intestine. 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Three-step hydroxylation of vitamin D 3 by a genetically engineered CYP10 5A1 Enzymes and catalysis Keiko Hayashi 1 , Kaori Yasuda 1 , Hiroshi