Báo cáo khoa học: Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1 doc

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:
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
().
Acknowledgement
This work was supported, in part, by a Ministry of
Education, Culture, Sports, Science and Technology
grant, and the Novozymes Japan Research Fund.
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