Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D 3 hydroxylase

Article abstract of DOI:10.1074/jbc.M110.147009

Vitamin D₃ hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D₃ (VD₃) to 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂VD ₃) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD₃, i.e. the conversion of VD₃ to 25-hydroxyvitamin D₃ (25(OH)VD₃) and then of 25(OH)VD₃ to 1α,25(OH) ₂VD₃, a hormonal form of VD₃. Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD₃-bound, and 25(OH)VD₃-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substratebound structure of Vdh-K1 accommodates both VD₃ and 25(OH)VD₃ but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD₃ hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD₃ 25-hydroxylase and 25(OH)VD₃ 1α-hydroxylase activities.

Full text of DOI:10.1074/jbc.M110.147009

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2010/07/27/M110.147009.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 41, pp. 31193–31201, October 8, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Structural Evidence for Enhancement of Sequential Vitamin
D3 Hydroxylation Activities by Directed Evolution of
□
S
Cytochrome P450 Vitamin D3 Hydroxylase*
Received for publication, May 23, 2010, and in revised form, July 17, 2010 Published, JBC Papers in Press, July 27, 2010, DOI 10.1074/jbc.M110.147009
Yoshiaki Yasutake‡, Yoshikazu Fujii§¶, Taiki Nishioka§, Woo-Kwang Cheon‡1, Akira Arisawa¶, and Tomohiro Tamura‡§2
From the ‡Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),
Sapporo 062-8517, the §Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, and ¶Bioresource Laboratories, Mercian Corporation, Shizuoka 438-0078, Japan
Vitamin D3 (VD3)3 is a B-ring opening secosteroid involved
in a wide variety of biological functions in mammals (1). In
humans, VD3 is converted into its physiologically active form,
1␣,25-dihydroxyvitamin D3 (1␣,25(OH)2VD3), via hydroxyla-
tion reactions that are catalyzed by several cytochrome P450s
(CYPs) (1, 2). The first hydroxylation is done at the C25 position
of VD3 by CYP27A1 (2, 3) and CYP2R1 (2, 4) in the liver to
produce 25-hydroxyvitamin D3 (25(OH)VD3). The second pro-
ceeds at the C1␣ position of 25(OH)VD3 by CYP27B1 in the
kidney (5) (Fig. 1). The final product, 1␣,25(OH)2VD3, func-
tions as a hormone with a critical role in maintaining calcium
and phosphate homeostasis as well as in controlling the differ-
entiation and proliferation of multiple cell types (1, 2, 6).
Indeed, the many symptoms associated with VD3 deficiency
and the VD metabolic disorder, which include psoriasis, osteo-
porosis, rickets, and hypoparathyroidism, are treated using
1␣,25(OH)2VD3 and its derivatives (1).
Vitamin D3 hydroxylase (Vdh) isolated from actinomycete
Pseudonocardia autotrophica is a cytochrome P450 (CYP)
responsible for the biocatalytic conversion of vitamin D3 (VD3)
to 1␣,25-dihydroxyvitamin D3 (1␣,25(OH)2VD3) by P. auto-
trophica. Although its biological function is unclear, Vdh is
capable of catalyzing the two-step hydroxylation of VD3, i.e. the
conversion of VD3 to 25-hydroxyvitamin D3 (25(OH)VD3) and
then of 25(OH)VD3 to 1␣,25(OH)2VD3, a hormonal form of
VD3. Here we describe the crystal structures of wild-type Vdh
(Vdh-WT) in the substrate-free form and of the highly active
quadruple mutant (Vdh-K1) generated by directed evolution in
the substrate-free, VD3-bound, and 25(OH)VD3-bound forms.
Vdh-WT exhibits an open conformation with the distal heme
pocket exposed to the solvent both in the presence and absence
of a substrate, whereas Vdh-K1 exhibits a closed conformation
in both the substrate-free and substrate-bound forms. The
results suggest that the conformational equilibrium was largely
shifted toward the closed conformation by four amino acid sub-
stitutions scattered throughout the molecule. The substrate-
bound structure of Vdh-K1 accommodates both VD3 and
25(OH)VD3 but in an anti-parallel orientation. The occurrence
of the two secosteroid binding modes accounts for the regio-
selective sequential VD3 hydroxylation activities. Moreover,
these structures determined before and after directed evolution,
together with biochemical and spectroscopic data, provide
insights into how directed evolution has worked for significant
enhancement of both the VD3 25-hydroxylase and 25(OH)VD3
1␣-hydroxylase activities.
Although the chemical synthesis of 1␣,25(OH)2VD3 from
cholesterol is an established method, it is inefficient, the maxi-
mum yield is no more than 1% (7). Alternatively, biocatalytic
conversion by the actinomycete Pseudonocardia autotrophica
is currently in practical use for the industrial production of
1␣,25(OH)2VD3 (8, 9). We have recently cloned the gene
encoding the VD3 hydroxylating enzyme (Vdh) of P. auto-
trophica, showing that Vdh is a CYP with ϳ43% amino acid
sequence similarities with the members of the CYP107 family
(10). There were two more interesting findings of this study.
First, we found that Vdh is capable of catalyzing regio- and
stereoselective sequential hydroxylations at the C25 and C1␣
positions of VD3. An in vitro assay using a recombinant enzyme
revealed that Vdh clearly exhibits VD3 25-hydroxylase and
25(OH)VD3 1␣-hydroxylase activities (Fig. 1). These enzymatic
properties are consistent with phenomena in the course of bio-
transformation: the 25(OH)VD3 level initially increases, fol-
lowed by a gradual increase in the 1␣,25(OH)2VD3 level in cul-
ture media. No 1␣-hydroxyvitamin D3 was detected in either in
vivo or in vitro experiments. Second, the VD3 25-hydroxylase
activity of Vdh is comparable with that of physiologically VD3-
* This work was supported in part by the Development of Basic Technologies
for Advanced Production Methods Using Microorganism Functions pro-
ject of the New Energy and Industrial Technology Development Organiza-
tion (NEDO) of Japan. Synchrotron radiation experiments were conducted
under the approval of 2007G650 at Photon Factory.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1, Figs. S1–S5, and Movies S1 and S2.
The atomic coordinates and structure factors (codes 3A4G, 3A4H, 3A4Z, 3A50,
and3A51)havebeendepositedintheProteinDataBank, ResearchCollabora-
tory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
1 Present address: Dept. of Physical Education, College of Physical Education,
Keimyung University, 2800 Dalgubeoldaero, Dalseo-gu, Daegu 704-701,
South Korea.
2 To whom correspondence should be addressed: Bioproduction Research 3 The abbreviations used are: VD3, vitamin D3; CYP, cytochrome P450; Vdh,
Institute, National Institute of Advanced Industrial Science and Technol-
ogy (AIST), 2-17-2-1, Tsukisamu-Higashi, Toyohira, Sapporo 062-8517,
Japan. Tel.: 81-11-857-8938; Fax: 81-11-857-8980; E-mail: t-tamura@
aist.go.jp.
vitamin D3 hydroxylase; 25(OH)VD3, 25-hydroxyvitamin D3; CD, cyclodex-
trin; PM␤CD, partially methylated ␤-cyclodextrin; BisTris, 2-[bis(2-hy-
droxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; r.m.s., root mean
square.
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Structures of P450 Vdh before and after Directed Evolution
EXPERIMENTAL PROCEDURES
Materials—All enzymes used for
genetic manipulation were pur-
chased from New England Biolabs,
Takara, Promega, and Stratagene.
Spinach ferredoxin and ferre-
doxin reductase used for enzyme
assay were purchased from Sigma.
25(OH)VD3 was kindly provided by
Mercian Co. Ltd. Partially methyl-
ated ␤-cyclodextrin (PM␤CD) was
purchased from Junsei Chemical
Inc. All other chemicals were pur-
FIGURE 1. Scheme of catalytic conversion of VD3 to 1␣,25(OH)2VD3. In humans, VD3 is physiologically
activated mainly by CYP27A1, CYP2R1, and CYP27B1 in the liver and kidney. Vdh from P. autotrophica also
catalyzes the conversion of VD3 to 1␣,25(OH)2VD3 by regio- and stereoselective sequential hydroxylations.
chased from Sigma or WAKO Pure
Chemicals Inc.
Protein Expression and Purifica-
metabolizing CYP27A1 (2, 3) and CYP2R1 (2, 4) despite the fact
that VD3 is probably a nonnative substrate for Vdh.
tion—The recombinant proteins used in this work contained a
His6 tag at the C terminus. Overproduction of Vdh-WT was
carried out using a Rhodococcus erythropolis expression system
(19) or E. coli, and purified by Ni-affinity chromatography and
anion exchange chromatography, as previously described (10,
20). The highly active Vdh-K1 mutant (T70R/V156L/E216M/
E384R) was generated by directed evolution (10). Recombinant
Vdh-K1 was produced using pET22b expression vector (Nova-
gen) in E. coli strain BL21(DE3). Overexpression was induced
by the addition of 0.1 mM isopropyl ␤-D-thiogalactoside for 20 h
at 22 °C in Luria-Bertani (LB) medium supplemented with 100
␮M FeSO4 and 80 ␮g/ml of 5-aminolevurinic acid. The cells
were harvested and resuspended in buffer A (50 mM Tris-HCl,
pH 7.5, and 10% glycerol) supplemented with 2 mM dithiothre-
itol (DTT). The cells were lysed by sonication, and the homo-
genate was clarified by centrifugation. The supernatant was
dialyzed against buffer A, and then applied to a Ni-affinity col-
umn (HIS-SELECT resin; Sigma) pre-equilibrated with buffer
A. The column was washed with buffer A, and the enzyme was
eluted with a linear gradient of 0–400 mM imidazole in buffer
A. The red fractions were collected, dialyzed against buffer B
(20 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM DTT, and 0.5 mM
EDTA), and subsequently applied to a DEAE-Sephacel anion-
exchange column (GE Healthcare) pre-equilibrated with buffer
B. The enzyme was eluted with a linear gradient of 0–400 mM
NaCl in buffer B. The individual fractions were analyzed by
SDS-PAGE. A carbon monoxide difference spectral assay was
performed to verify the Vdh concentration (21). Vdh single
mutants (T70R, V156L, E216M, and E384R) were also prepared
using inverse PCR (22) with appropriate synthetic oligonucleo-
tides as primers and pET29-Vdh-WT (10) as the template. Each
mutant was expressed by E. coli BL21(DE3) and purified with
the same procedure as for Vdh-K1.
CYPs are hemoproteins found across almost all of the Trees
of Life and have extremely diverse biological functions, includ-
ing biosynthesis of steroid hormones, lipids, and complex anti-
biotics, such as polyketides, detoxification of xenobiotics, drug
metabolism, and bioconversion of recalcitrant molecules into
usable carbon sources (11). A typical reaction catalyzed by
CYPs is hydroxylation (monooxygenation), although several
CYPs catalyze even more complex reactions such as epoxida-
tion, C-C bond coupling and cleavage, and dehalogenation.
This intriguing functional diversity of CYPs has led to many
studies on their three-dimensional structures, which attempt to
understand the structural mechanism of substrate recognition,
interactions with redox partner proteins, electron and proton
transfer, and catalytic reactions (12). Additionally, the versatil-
ity of CYPs is highly attractive for their potential uses as bio-
catalysts in chemical and pharmaceutical applications. Further-
more, protein engineering has been applied to improve and
specialize the function of CYPs from bacteria and mammals
(13–18). To realize more effective bioconversion of VD3, bio-
transformation activity from VD3 to 25(OH)VD3 was improved
by a combination of random and site-saturated mutagenesis,
and finally, a highly active Vdh mutant (Vdh-K1) was obtained
(10). Vdh-K1 is a quadruple mutant (T70R/V156L/E216M/
E384R) that is ϳ22 times more active in the hydroxylation of
VD3 to 25(OH)VD3 than wild-type Vdh (Vdh-WT) in in vivo
bioconversion using Escherichia coli cells (10). Intriguingly, the
four mutational points do not lie in the substrate-binding
pocket, but are scattered throughout the molecule. Thus, it is of
considerable interest as to how these mutations effect the sig-
nificant enhancement in Vdh activity and how the enzyme rec-
ognizes each substrate.
Here we report the crystal structures of Vdh-WT and
Vdh-K1 in the presence and absence of VD3/25(OH)VD3. The
directed evolution and the x-ray structures show the impor-
tance of the conformational transition from open to closed
states rather than the local active-site structure for activity
enhancement. Furthermore, the VD3- and 25(OH)VD3-bound
Vdh-K1 structures shed light on the occurrence of two anti-
parallel substrate-binding modes, reliably enabling regioselec-
tive sequential hydroxylation of VD3.
UV-visible Spectral Measurements—The substrate-induced
spectral shift was monitored using a JASCO V-630 biospectro-
photometer with 1-cm path length quartz cells. VD3 and
25(OH)VD3 were dissolved in dimethyl sulfoxide at concentra-
tions ranging from 50 ␮M to 2 mM, and 10 ␮l of the substrate
solution was added to 990 ␮l of reaction mixture containing 2.5
␮M Vdh-WT or Vdh mutants, 20 mM Tris-HCl, pH 7.5, and
0.05% PM␤CD or 0.02% ␥-cyclodextrin (␥CD). PM␤CD and
31194 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structures of P450 Vdh before and after Directed Evolution
␥CD were used to increase the water solubility of VD3 and bic crystals were grown in space group P212121 with unit cell
25(OH)VD3, respectively (9, 23). CD concentrations referred to parameters a ϭ 63.6, b ϭ 65.8, c ϭ 102.3 Å in the presence of
those adopted in actual VD3 bioconversion by P. autotrophica. saturated 25(OH)VD3. Vdh-K1 was crystallized in orthorhom-
The correct Kd values cannot be determined due to the low bic space group P212121 with unit cell dimensions of a ϭ 77, b ϭ
substrate solubility and the presence of inclusion complex of 172, c ϭ 189 Å, both in the presence and absence of
substrate and CD.
VD3/25(OH)VD3.
Enzyme Activity Measurements—VD3 25-hydroxylase and
Structure Determination and Model Analysis—The structure
25(OH)VD3 1␣-hydroxylase activities were measured by of Vdh-WT in trigonal crystal form was determined by the
reconstitution experiments. One milliliter of reaction mixture molecular replacement method with the program AMoRe (25),
containing 500 nM Vdh-WT or 100 nM Vdh-K1, 81.4 mM Tris- using P450 EryF model fragments (PDB code 1JIN) (26) as
HCl, pH 7.5, 100 mM NaCl, 96 ␮g/ml of spinach ferredoxin, 0.1 search probes. The structures of Vdh-WT and Vdh-K1 in
units/ml of spinach ferredoxin reductase, 3 units/ml of glucose orthorhombic crystal forms were solved by molecular replace-
dehydrogenase, 2 mM NADH, 2 mM NADPH, 60 mM D-glucose, ment with the program MOLREP (27), using the trigonal
1% dimethyl sulfoxide, and 30 ␮M VD3 or 25(OH)VD3 was used Vdh-WT structure as a search model. All models were refined
for enzyme activity measurements. Enzymatic reaction was ini- using the program REFMAC5 (28), and were inspected and
tiated by the addition of NAD(P)H, followed by incubation at corrected in the graphic program COOT (29). A randomly
30 °C. Aliquots of the reaction mixture were collected after 20 selected 5% of the observed reflections were set aside for cross-
(Vdh-WT) or 5 min (Vdh-K1), and extracted once with 1.5 ml validation analysis and used to monitor various refinement
and once with 0.75 ml of ethyl acetate. The combined ethyl stage. VD3 and 25(OH)VD3 models in Vdh-K1 were built based
acetate phase was allowed to dry, and the resulting residue was on a Fo Ϫ Fc difference Fourier map in COOT. Electron density
dissolved in 200 ␮l of methanol. The methanol solution was for 25(OH)VD3 is clear, whereas that for VD3 is partly ambig-
analyzed by a high-performance liquid chromatography uous due to the high mobility and high temperature factors.
(HPLC) apparatus equipped with a J’sphere ODS-H80 (I.D. The validities of all substrate models were verified by calculat-
4.6 ϫ 75 mm; YMC, Japan) controlled at a column temperature ing the simulated annealing 2Fo Ϫ Fc composite omit map with
of 40 °C. Samples were resolved on the column using a linear the program CNS 1.1 (30) (supplemental Fig. S4). The stereo-
gradient of 50 to 100% acetonitrile for 12 min, followed by elu- chemical qualities of the final refined models were assessed by
tion with 100% acetonitrile for 13 min at a flow rate of 1.5 the program PROCHECK (31). The x-ray data collection and
ml/min. The metabolites were detected by UV at 265 nm.
refinement statistics are summarized in Table 1. Structure
Crystallization and X-ray Diffraction Studies—Purified Vdh- superposition and assessment of conformational changes with
WT was concentrated to 20 mg/ml in 20 mM Tris-HCl, pH 7.5, difference distance matrices were performed with the program
and 0–0.2% PM␤CD (for VD3 complex) or 0–0.2% ␥CD (for SuperPose (32). Molecular drawings were prepared using the
25(OH)VD3 complex), using a centrifugal ultrafiltration device program PyMOL (33).
with a 30-kDa cutoff membrane (Millipore). Vdh-K1 was also
RESULTS
concentrated to 20 mg/ml under the same conditions as Vdh-
WT, but supplemented with 20 mM NaCl. For substrate
Spectroscopic Characterization of Vdh—The binding affini-
complex formation, saturated VD3/25(OH)VD3 dissolved in ties of VD3/25(OH)VD3 to Vdh-WT and Vdh-K1 were exam-
dimethyl sulfoxide (ϳ100 mM) was added to each sample at the ined by UV-visible spectroscopy (Fig. 2). A small amount of
volume ratio 1:99, and incubated overnight at 4 °C. The crystal- PM␤CD or ␥CD were added to the assay mixture to dissolve
lization conditions were screened with 96-well sitting-drop VD3 or 25(OH)VD3, respectively (see “Experimental Proce-
plates at 20 °C by the sparse matrix approach. Optimization of dures”). The absorption spectra of Vdh-WT showed a Soret
the hit conditions were carried out by the hanging-drop vapor peak at 420 nm both in the presence and absence of VD3 and
diffusion technique in 24-well plates at 20 °C. Vdh-WT crystals 25(OH)VD3 (Fig. 2A). This indicates that Vdh-WT is not
were grown using reservoir solution containing 0.1 M BisTris, affected by substrate addition and the heme iron is present con-
pH 7.5, 50 mM CaCl2, 40–120 mM NaCl or KCl, and 32–40% sistently in a water-coordinated low-spin state. In contrast to
PEG 400 or PEG 550 monomethyl ether or 20% PEG 1000. Vdh-WT, significant spectral changes were detected for Vdh-
Vdh-K1 was crystallized using reservoir solution containing K1. Purified Vdh-K1 is present as a mixture of high-spin (Soret
0.1 M calcium acetate and 10–14% PEG 3350. Crystals were flash- peak at Ϸ390 nm) and low-spin (Soret peak at Ϸ420 nm) states,
cooled under a nitrogen gas stream at 100 K for x-ray diffraction and the ratio of high/low-spin states is very sensitive to the
studies. Prior to flash cooling, crystals of Vdh-K1 were short- addition of CDs. Typically, Vdh-K1 is present primarily as a
soaked with cryoprotectant, which is the crystallization mother low-spin state in the presence of PM␤CD, whereas it prefers a
liquor supplemented with 20% glycerol. All diffraction data high-spin state in the presence of ␥CD. Spectral titration of
were collected at the Photon Factory (PF, Tsukuba, Japan), Vdh-K1 with VD3 showed a spectral shift from the low-spin to
using the charge-coupled device detector (ADSC). The data high-spin state, which is a typical substrate-binding spectral
were indexed, integrated, and merged using the HKL2000 pro- change (type I spectral change; Fig. 2B). Unexpectedly, spectral
gram package (24). Vdh-WT was crystallized in two crystal titration of Vdh-K1 with 25(OH)VD3 produced a spectral shift
forms: the trigonal crystals were obtained in space group P31 from the high-spin (389 nm) to low-spin (414 nm) state (reverse
with unit cell parameters a ϭ b ϭ 62, c ϭ 99 Å, both in the type I spectral change), suggesting that an unidentified ligand is
presence and absence of VD3/25(OH)VD3, and the orthorhom- coordinated to the heme iron in the presence of 25(OH)VD3
OCTOBER 8, 2010•VOLUME 285•NUMBER 41
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Structures of P450 Vdh before and after Directed Evolution
overall structure of Vdh exhibits a typical triangular-shaped
P450-fold consisting of 13 ␣-helices (A-helix to M-helix) and 8
␤-strands (a-strand to h-strand). The heme cofactor is located
at the center of the molecule and is sandwiched between distal
I-helix and proximal L-helix. The heme propionate groups are
tightly bound to side chains Arg289, His95, and His345. The thiol
group of Cys347 is coordinated to the heme iron, as shown in
previously reported CYP structures. A sequence similarity
search indicated that Vdh is a member of the family CYP107.
The structures of three molecular species of CYP107 (EryF (26,
35, 36), PikC (37, 38), and BioI (39)) have been solved to date; a
pairwise structure comparison revealed that Vdh is the most
related to PikC (CYP107L1).
Vdh-WT Forms an Open Conformation—The crystal struc-
ture of Vdh-WT was determined in the substrate-free form at a
resolution of 1.75 Å (Fig. 3). The asymmetric unit comprises
one Vdh-WT monomer. Vdh-WT is most structurally related
to substrate-free PikC in the open conformation (PDB code
2BVJ, chain B) (37) with a root mean square deviation (r.m.s.
deviation) of 2.6 Å for 376 C␣ atoms (supplemental Fig. S3).
Differences in the main chain conformation are observed in two
loop regions: an N-terminal loop (residues 10–17) and a loop
between B-helix and C-helix commonly referred to as a “BC-
loop” (residues 71–85). The BC-loop is known to be flexible
and is partially disordered in the substrate-free PikC structure.
The BC-loop in Vdh-WT protrudes from the globular body of
the P450-fold, and the substrate-binding pocket on the distal
side of the heme is widely open and exposed to the solvent (Fig.
3 and supplemental Fig. S5A). The water ligand is bound to the
distal side of the heme iron with a Fe-O distance of 2.4 Å. Extra
electron density is also observed in the large pocket mainly
surrounded by hydrophobic residues, in which a model of
polyethylene glycol (PEG) was reasonably fitted. The bound
PEG is likely a crystallization artifact.
We also obtained crystals of Vdh-WT in the presence of sub-
strates in two different forms. The same trigonal crystals were
grown with saturated VD3 or 25(OH)VD3, but the structures
were completely similar to that in the substrate-free open con-
formation. Vdh-WT also crystallized in the orthorhombic
space group P212121 with saturated 25(OH)VD3, and the struc-
ture was determined at 3.05-Å resolution. The overall structure
in the orthorhombic crystal also superposed very well onto that
in trigonal form, including the BC-loop, with an r.m.s. deviation
of 1.0 Å for 395 C␣ atoms (supplemental Fig. S2A). In both
structures, no electron density for VD3/25(OH)VD3 bound to
the active-site pocket was observed. These results indicate that
the open conformation of Vdh-WT is stable and not affected by
crystal lattice forces, even in the presence of saturated
FIGURE 2. Spectral changes of Vdh-WT and Vdh-K1 with VD3/25(OH)VD3.
UV-visible spectra for Vdh-WT on addition of 0, 10, and 20 ␮M VD3/25(OH)VD3
(panel A), and for Vdh-K1 on addition of 0–20 ␮M VD3 (panel B) and 0–20 ␮M
25(OH)VD3 (panel C). Arrows indicate the directions of spectral changes in
response to the increase in VD3/25(OH)VD3 concentrations. Wavelength (nm)
at maximum height of Soret band observed in each spectral measurement is
also indicated.
(Fig. 2C). The ligand exhibiting the Soret peak at 414 nm was VD3/25(OH)VD3.
identified as the C3 OH group (3␤-OH) of 25(OH)VD3 by x-ray
Closed Structure of Vdh-K1 in Substrate-free and Substrate-
bound Forms—The structures of Vdh-K1 in the substrate-free,
structure analysis, as described later.
General Description of Vdh Structure—The crystal structures VD3-bound, and 25(OH)VD3-bound forms were determined at
of Vdh-WT in the substrate-free form and Vdh-K1 in both the 2.3-, 2.05-, and 2.0-Å resolutions, respectively (Fig. 3). Crystal-
substrate-free and substrate-bound forms were determined by lization screening of Vdh-K1 yielded only one orthorhombic
molecular replacement using P450 EryF (CYP107A1) as a form with relatively large unit-cell dimensions compared with
search model (Fig. 3 and Table 1). Molecules in all crystal forms Vdh-WT crystals. The asymmetric unit contains 5 Vdh-K1
have a well defined continuous electron density, except for sev- monomers that are not related by point-group symmetry. The
eral N-terminal residues and C-terminal His tag regions. The structures of all the refined models of Vdh-K1 (15 monomers)
31196 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structures of P450 Vdh before and after Directed Evolution
FIGURE 3. Ribbon diagram of overall structure of Vdh-WT (open form) and Vdh-K1 (closed form). Overlay of these two structures is also shown. The heme
cofactor is in the sphere, and the bound-substrate (25(OH)VD3) and the side chains of mutational residues at positions 70, 156, 216, and 384 are in sticks. The
␣-helices are labeled alphabetically from the N to C terminus.
TABLE 1
Crystallographic parameters and refinement statistics
PDB code
3A4Z
3A4G
3A4H
3A50
3A51
Model
Vdh-WT
Vdh-WT
Vdh-K1
Vdh-K1
Closed
5
Vdh-K1
Closed
5
Conformation
No. monomers in the AU
Substrates
Open
Open
Closed
1
1
5
Substrate-free
Substrate-free
Substrate-free
VD3
25(OH)VD3
Data collection
Beamline
PF-BL5A
1.0000
PF-BL17A
1.0000
AR-NE3A
1.0000
AR-NE3A
1.0000
AR-NW12A
1.0000
Wavelength (Å)
Space group
P31
P212121
P212121
P212121
P212121
Unit cell parameters (Å, deg)
61.7, 61.7, 98.8,
90, 90, 120
50–1.75 (1.81–1.75)
42,012
63.6, 65.8, 102.3,
90, 90, 90
77.4, 172.5, 189.9,
90, 90, 90
77.4, 172.3, 189.1,
90, 90, 90
77.2, 171.8, 189.1,
90, 90, 90
Resolution range (Å)a
Unique reflections
Redundancya
50–3.05 (3.16–3.05)
8,366
50–2.20 (2.24–2.20)
129,009
50–2.05 (2.09–2.05)
158,815
50–2.00 (2.07–2.0)
170,383
5.3 (3.2)
3.6 (3.8)
7.3 (6.0)
7.5 (6.3)
7.4 (7.0)
Completeness (%)a
I/␴ (I)a
99.3 (93.2)
21.9 (3.7)
0.075 (0.316)
23.2
97.6 (97.9)
18.9 (6.0)
100.0 (99.8)
26.0 (2.9)
100.0 (99.8)
26.9 (2.9)
100.0 (100.0)
32.5 (3.3)
a,b
Rmerge
0.090 (0.317)
45.0
0.101 (0.500)
27.9
0.107 (0.540)
23.3
0.077 (0.594)
29.5
Wilson B-factor (Å2)
Refinement
c
Rwork/Rfree
0.185/0.236
0.015
0.217/0.270
0.006
0.201/0.244
0.015
0.201/0.240
0.013
0.197/0.234
0.014
R.m.s.deviation bond lengths (Å)
R.m.s.deviation bond angles (deg)
Total atoms
1.53
0.96
1.56
1.50
1.55
3,451
3,138
16,621
16,977
17,293
Average B-factors (Å2)
Overall
28.7
44.1
34.1
28.9
30.4
Ligands
35.3 (PEG)
69.0 (VD3)
41.3 (25(OH)VD3)
90.6/9.4/0.0/0.0
Ramachandran plot (%)
89.6/10.4/0.0/0.0
89.5/10.5/0.0/0.0
91.1/8.9/0.0/0.0
90.6/9.4/0.0/0.0
a Data for the highest resolution shell are provided in parentheses.
b Rmerge ϭ ⌺h ⌺i͉Ih,i Ϫ ͗Ih͉͘/⌺h⌺i Ih,i, where ͗Ih͘ is the mean intensity of a set of equivalent reflections.
c Rwork ϭ ⌺͉Fobs Ϫ Fcalc͉/⌺ Fobs for the 95% of the reflection data used in the refinement. Fobs and Fcalc are observed and calculated structure factor amplitudes, respectively. Rfree
is the equivalent of Rwork, except that it was calculated for a randomly chosen 5% test set excluded from the refinement.
are almost identical (r.m.s. deviation, Ͻ 0.7 Å), and no relatively additional conformational change occurs in a loop connecting
large structural differences were observed, suggesting that the H- and I-helix (HI-loop), and induces the extension of H-helix
conformation of Vdh-K1 is also stable even in the substrate- by 4 residues (Fig. 4). Similar conformational changes were also
free, VD3-bound, and 25(OH)VD3-bound states. Notably, local observed in the PikC structure, and Vdh-K1 is more structur-
structural differences were found between Vdh-WT and Vdh-K1 ally related to the closed form of PikC (PDB code 2BVJ, chain A;
(Figs. 3, 4, supplemental Fig. S2B, and Movies S1 and S2). The r.m.s. deviation of 3.0 Å for 378 C␣ atoms) than to the open
differences were most evident in the repositioning of the F- and form of PikC (PDB code 2BVJ, chain B; r.m.s. deviation of 4.0 Å
G-helices, which are shifted by a distance of up to 8 Å, and the for 378 C␣ atoms; supplemental Fig. S3) (37). The BC-loop is
loop connecting the F- and G-helix (FG-loop) contacts the known to often move toward the substrate-binding pocket in
opposite loop (C-terminal loop) via two hydrogen bonds. This response to substrate binding, whereas it is motionless in Vdh
motion leads to a closed conformation where the substrate- (Fig. 3). Therefore, the substrate-binding pocket is not fully
binding pocket is covered by FG-helices (Figs. 3 and 4). An closed and is partially accessible by the solvent. The four muta-
OCTOBER 8, 2010•VOLUME 285•NUMBER 41
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Structures of P450 Vdh before and after Directed Evolution
and the CD-rings of VD3 are surrounded by hydrophobic side
chain atoms of residues Met86, Ile88, Leu89, Leu171, Ile235
,
Leu232, Pro287, and Leu387 with distances ranging from 3.6 to
4.5 Å, whereas the A-ring moiety is considerably exposed to the
solvent (Fig. 5B). Although Trp67, Pro83, Lys180, Asn181, and
Met184 derived from BC-loop, FG-loop, and G-helix lie near
the A-ring, there are no specific interatomic interactions
with a unique hydrophilic 3␤-OH of A-ring. In contrast, the
electron density for 25(OH)VD3 is very clear, and it is evi-
dently bound in an anti-parallel orientation (Fig. 5, C and D, and
supplemental Fig. S4). The mean B-factor value for 25(OH)VD3
atoms is 41 Å2, which is lower than that for VD3. The clear
electron density also revealed that A-ring forms in a boat con-
formation with an axial 3␤-OH occupying the sixth coordinate
position of the heme iron with a mean distance of 2.5 Ϯ 0.1 Å.
The hydroxylating site C1 is located at a distance of 5.0 Ϯ 0.0 Å
from the heme iron. The 3␤-OH coordination to the heme iron
explains the Soret peak at 414 nm in the spectroscopic data.
Unlike the A-ring of VD3, the aliphatic side chain of
25(OH)VD3 does not fluctuate even though it is partly exposed
to the solvent and can be immobilized alongside of the FG-loop
via van der Waals forces. In addition, in 4 of 5 molecules, hydro-
gen bonds form between 25-OH and one of the solvent mole-
cules clustered at the entrance of the substrate-binding pocket;
these also contribute to the attachment of the aliphatic side
chain.
DISCUSSION
Interpretation of Spectroscopic Data—The absorption spec-
tra of Vdh-WT show that the heme iron is present in a water-
liganded low-spin state both in the presence and absence of a
FIGURE4. StereoviewC␣traceoftheVdh-WT(panelA)andVdh-K1(panel
B) in the region composing C-, FG-, and HI-helices and the C-terminal
loop. Mutationalresiduesatpositions156, 216, and384aredepictedin sticks.
Salt bridges and hydrogen bonds broken and reconstituted during open/
close motion are shown as dashed lines. Side chains involving these hydro- substrate (Fig. 2A). The crystal structures of Vdh-WT also
philic interactions or hydrophobic residues nearby Leu156/Val156 creating
reveals that no substrate is bound to Vdh-WT even in the pres-
hydrophobic core are in thin sticks and labeled.
ence of saturated VD3/25(OH)VD3. These results clearly show
a very low substrate binding affinity despite the fact that the
tional positions in Vdh-K1 (T70R, V156L, E216M, and E384R) catalytic activity of Vdh-WT is comparable with those of phys-
are not located at the substrate-binding site but are scattered iologically VD3-metabolizing CYP27A1 and CYP2R1 (2–4).
throughout the molecule. We discuss later how these muta- One possible explanation is that substrate binding to Vdh-WT
tions are involved in the open/closed conformational change may occur depending on the association with a redox partner
(see “Discussion”).
protein. Several cases have been reported to date where a redox
VD3 and 25(OH)VD3 Are Bound to Vdh-K1 in an Anti-par- protein functions as an effector that promotes substrate bind-
allel Orientation—Vdh catalyzes the two-step hydroxylation of ing to CYPs (40, 41). In contrast to Vdh-WT, Vdh-K1 exhibits
VD3 to 1␣,25(OH)2VD3 via 25(OH)VD3. Because the hydroxy- spectral changes in response to the addition of VD3/
lation positions are discrete, VD3 and 25(OH)VD3 must be 25(OH)VD3 (Fig. 2, B and C). The absorption peak in the Soret
bound in different orientations. The electron density shows band is shown at 393 nm with VD3 and at 414 nm with
that VD3 is bound in an extended conformation at the active- 25(OH)VD3. The former clearly corresponds to a five-coordi-
site pocket, and the aliphatic side chain including C25 is located nate high-spin state of the heme iron caused by VD3 binding,
in the vicinity of the heme iron (Fig. 5, A and B). The mean whereas the latter indicates a six-coordinate low-spin state. The
distance between the C25 of VD3 and the heme iron of each structure of Vdh-K1 with bound 25(OH)VD3 revealed that the
molecule is 4.6 Ϯ 0.1 Å. No solvent is coordinated to the distal 3␤-OH group of 25(OH)VD3 is a sixth coordination ligand that
site of the heme iron probably due to VD3 binding. These obser- is positioned very close to the heme iron. Ligation of the OH
vations support the high-spin state of the heme iron as shown group to the heme iron was previously reported in the structure
by spectroscopic data. The mean refined B-factor value for VD3 of P450cam complexed with the product 5-exo-hydroxycam-
atoms is 69 Å2, suggesting that VD3 is not tightly bound to the phor (42). In addition, several mammalian CYPs show the
enzyme. Actually, the conformation of the five refined VD3 reverse type I spectral change in response to the addition of a
models in each monomer is varied, especially in the A-ring moi- substrate or inhibitor carrying OH groups (Ref. 43 and refer-
ety for which the electron density is even lower and unclear (Fig. ences therein). Obviously, the OH-bound heme iron reduces
5, A and B, and supplemental Fig. S4). The aliphatic side chain the catalytic activity of CYP, because the low-spin ferric state
31198 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 41•OCTOBER 8, 2010

Structures of P450 Vdh before and after Directed Evolution
hydrophobic cluster formed by the
side chains of Phe164, Leu191, Leu194
,
Leu154, Leu207, and Tyr190 (Fig. 4A).
The substitution from Val to Leu
gives rise to steric hindrance with
the side chains Phe164 and Tyr190 in
the open state, thus requiring the
spatial repositioning of these hydro-
phobic side chains. In the Vdh-K1
structure, the hydrophobic cluster
is actually rearranged by changing
the torsion geometries of these side
chains together with the approxi-
mately ϳ8-Å shift of FG-helices,
and consequently, the interatomic
distances are reoptimized (Fig. 4B).
Glu216 is located at the protrusive
HI-loop, and its side chain is fully
exposed to the solvent with a rela-
tively high B-factor value (about
55 Å2) in the Vdh-WT structure.
The solvent-exposed methionine is
unfavorable for protein stability,
and the E216M mutation possibly
functions as a driving force for
conformational change of the HI-
loop and extension of H-helix
involving Met216. It is of interest to
note that several salt bridges and
hydrogen bonds are broken and
reconstructed between residues
derived from the C-, FG-, and HI-
helices region during the con-
formational change (Fig. 4). These
FIGURE 5. VD3 and 25(OH)VD3 binding to Vdh-K1. Final 2Fo Ϫ Fc electron density map for VD3 (panel A) and
25(OH)VD3 (panel C) contoured at 0.8␴ level (chain ID, A). Unbiased simulated annealing composite omit map
for all chains in the asymmetric unit are shown in supplemental Fig. S4. B, stereo view active-site structure with
bound VD3 in chain A. All five refined models of VD3 in each chain in the asymmetric unit are superimposed and
shown as thin sticks. Residues creating the substrate-binding pocket are shown in sticks and are labeled. The
heme cofactor is the sphere. D, stereo view active site structure with bound 25(OH)VD3 prepared in the same
way and in the same orientation to panel B.
with very low redox potential precludes its reduction of the rearrangements of interatomic interactions imply that motions
heme iron that is needed for the subsequent electron transfer in the FG- and HI-helices regions are mutually cooperative. In
and dioxygen binding. Therefore, further repositioning of the contrast, the two other mutations (E384R and T70R) lie in the
A-ring of 25(OH)VD3 would be required for promoting the structurally rigid region. Arg384 is located at the C-terminal
reduction and monooxygenation at the C1␣ site.
loop that interacts with the FG-loop in the closed Vdh-K1
The spectral data suggest that the binding affinities of both structure. The main chain O of Arg384 and side chain OH of
substrates to Vdh increase by directed evolution. The specific Ser385 form hydrogen bonds to the main chain N of Asp174 and
activity measurements also showed that Vdh-K1 is ϳ12 times main chain O of Val172 at the FG-loop, respectively. In addition,
more active for VD3 25-hydroxylation and 25 times more electrostatic interactions occur between the side chain guanidyl
active for 25(OH)VD3 1␣-hydroxylation than Vdh-WT (sup- of Arg384 and carboxyl of Asp174 with distances of 3.5–5 Å (Fig.
plemental Table S1). The high binding affinity and catalytic 4B). It is thus likely that substitution from acidic to basic resi-
activity of Vdh-K1 for 25(OH)VD3 are totally unexpected, dues at position 384 somewhat increases the stability of the
because we monitored only the VD3 25-hydroxylase activity in closed conformation. It is unclear whether Arg70 contributes
the directed evolution experiments. These results strongly sug- to the conformational change and increase in substrate bind-
gest that the four mutations do not improve the substrate spec- ing affinity. In several CYPs, arginine residues at the
ificity but affect the underlying enzyme mechanisms that are entrance of the substrate access channel are suggested to
common to both hydroxylation activities.
play a crucial role in substrate recognition and/or guiding
Structural Insights into Activity Enhancement by Directed substrate entry and egress (44, 45). Likewise, Arg70 in
Evolution—Current structural studies reveal that the confor- Vdh-K1 is located at the entrance of the substrate-binding
mational change from the open to closed state results from the pocket and may provide a weak primary binding site that
four amino acid substitutions generated by directed evolution. improves the efficiency of substrate entry and egress. To
Then the question arises as to how these individual mutations investigate which mutations contribute most to the confor-
contribute to the conformational change. Val156 lies at the loop mational change, the absorption spectra of each single
connecting E- and F-helix, and its side chain is buried inside the mutant (T70R, V156L, E216M, and E384R) were also mea-
OCTOBER 8, 2010•VOLUME 285•NUMBER 41
JOURNAL OF BIOLOGICAL CHEMISTRY 31199

Structures of P450 Vdh before and after Directed Evolution
sured on addition of 0–20 ␮M substrates. As shown in iron and the A-ring containing the hydrophilic 3␤-OH group
supplemental Fig. S1, only the V156L mutant showed an obvious toward the entrance of the substrate-binding pocket exposed to
but relatively small substrate-induced spectral change, whereas the solvent (Fig. 5B). This binding orientation is possibly a con-
no spectral changes were observed for three other mutants sim- sequence of the need to prevent exposure of the hydrophobic
ilar to Vdh-WT. The results suggest that the combination of aliphatic side chain to the solvent, and is also consistent with
these mutations is essential to elicit high substrate binding the fact that Vdh exhibits only 25-hydroxylase activity against
affinities observed in Vdh-K1.
VD3. Once C25 is hydroxylated, 25(OH)VD3 is bound to the
Elaborate rearrangements of interatomic interactions enzyme in an anti-parallel orientation with 3␤-OH of A-ring
around CFGHI helices imply that the closed structure observed anchored to the heme iron, thereby enabling the subsequent
in Vdh-K1 is not a novel conformational state that resulted 1␣-hydroxylation. The existence of two substrate binding
from directed evolution but rather a naturally pre-existing modes in different orientations implies that Vdh-K1 is not
state. The original residues at mutational positions seemingly functionally specialized.
cause no serious problems even in the closed conformation.
In mammals, VD3 is hydroxylated by mainly CYP27A1,
Furthermore, open Vdh-WT and closed Vdh-K1 are structur- CYP2R1, and CYP27B1. Of these CYPs, the crystal structure
ally related to the naturally existing open and closed states of of CYP2R1 has been determined in complex with VD3 (4). In
wild-type PikC (supplemental Fig. S3). Several CYPs are known CYP2R1, VD3 is fully buried in the hydrophobic active site
to undergo conformational changes even in the absence of a pocket, and the A-ring of VD3 facing the protein surface is also
substrate, and the substrate complex formation likely arises surrounded and stabilized by hydrophobic interactions with
from a pre-existing conformational equilibrium rather than an several of the aromatic side chains (supplemental Fig. S5D).
induced fit mechanism (37, 46–48). In the present work, it is The well fitted substrate-bound form of CYP2R1 exemplifies
likely that the directed evolution was responsible for the shift in the functionally specialized structure of a VD3 25-hydroxylase.
conformational equilibrium toward the closed conformation Recently, the structures of P450 SU-1 (CYP105A1) from Strep-
and facilitation of VD3/25(OH)VD3 binding. Vdh-WT may sur- tomyces griseolus have been reported, which is another example
mount an energy barrier and transform from the open to closed of bacterial CYP that is capable of hydroxylating VD3 (34, 45).
state in response to the native substrate binding, and the cur- The product 1␣,25(OH)2VD3 lies sideways within the large
rent Vdh-K1 structure may mimic the putative closed form of active site pocket of CYP105A1, and the hydroxylating sites,
Vdh-WT when bound to the native substrate. Many CYPs show C25 and C1, are located at a distance of about 11 Å from the
extremely broad substrate specificity, presumably involving an heme iron (supplemental Fig. S5C). The authors suggested that
open/close motion (49). Typically, the open and closed struc- the 1␣,25(OH)2VD3 binding site far from the heme iron is pre-
tures correspond to the substrate-free and substrate-bound sumably a primary binding site, and the substrate would be
forms, respectively, and our results thus introduce the possibil- required to move closer to the heme iron for a catalytic reaction
ity that identification of mutational positions inducing a shift in (34, 45). These different VD3 binding conditions give a glimpse
conformational equilibrium toward the closed form would uns- into the impressive versatility of CYPs.
electively increase the substrate binding affinity and catalytic
activities.
Acknowledgments—We thank the entire beamline staff at Photon
Factory for technical support in the collection of x-ray diffraction
We observe a remarkable parallel between our directed evo-
lution studies of Vdh and natural selection of CYP51 mutants data.
identified in Candida albicans azole-resistant isolates that
emerged under selective pressure of antifungal drug flucon-
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