25-HydroxyvitaminD3 Synthesis by Enzymatic Steroid Side-Chain Hydroxylation with Water

Article abstract of DOI:10.1002/anie.201510331

The hydroxylation of vitaminD₃ (VD₃, cholecalciferol) side chains to give 25-hydroxyvitaminD₃ (25OHVD₃) is a crucial reaction in the formation of the circulating and biologically active forms of VD₃. It is usually catalyzed by cytochrome P450 monooxygenases that depend on complex electron donor systems. Cell-free extracts and a purified Mo enzyme from a bacterium anaerobically grown with cholesterol were employed for the regioselective, ferricyanide-dependent hydroxylation of VD₃ and proVD₃ (7-dehydrocholesterol) into the corresponding tertiary alcohols with greater than 99 % yield. Hydroxylation of VD₃ strictly depends on a cyclodextrin-assisted isomerization of VD₃ into preVD₃, the actual enzymatic substrate. This facile and robust method developed for 25OHVD₃ synthesis is a novel example for the concept of substrate-engineered catalysis and offers an attractive alternative to chemical or O₂ /electron-donor-dependent enzymatic procedures.

Full text of DOI:10.1002/anie.201510331

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Communications
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International Edition: DOI: 10.1002/anie.201510331
German Edition: DOI: 10.1002/ange.201510331
Biocatalysis
2
5-Hydroxyvitamin D Synthesis by Enzymatic Steroid Side-Chain
3
Hydroxylation with Water
Markus Warnke, Tobias Jung, Juri Dermer, Karin Hipp, Nico Jehmlich, Martin von Bergen,
Sascha Ferlaino, Alexander Fries, Michael Müller, and Matthias Boll*
Dedicated to Georg Fuchs on the occasion of his 70th birthday
Abstract: The hydroxylation of vitamin D (VD , cholecalci-
also to cardiovascular diseases, immunodeficiency, and dia-
3
3
[2]
ferol) side chains to give 25-hydroxyvitamin D (25OHVD ) is
betes. 25OHVD (2) represents the circulating form of VD ,
3
3
3
3
a crucial reaction in the formation of the circulating and
and its plasma concentration has been associated with VD -
3
[
3]
biologically active forms of VD . It is usually catalyzed by
linked disorders.
Classical chemical 25OHVD3 (2) synthesis procedures
3
cytochrome P450 monooxygenases that depend on complex
electron donor systems. Cell-free extracts and a purified Mo
enzyme from a bacterium anaerobically grown with choles-
terol were employed for the regioselective, ferricyanide-depen-
dent hydroxylation of VD and proVD (7-dehydrocholesterol)
[4]
involve multiple steps with low yields and specificities,
although recently more convenient procedures, for example,
methods involving anchoring on a solid phase, have been
[5]
developed. For enzymatic synthesis, CYPs either from liver,
3
3
[
6]
into the corresponding tertiary alcohols with greater than 99%
fungi, or actinomycetes have been employed. However, the
dependence on complex electron-donor systems consisting of
NAD(P)H, ferredoxin (Fd), and ferredoxin reductase (FdR)
has limited biotechnological applications; promising
approaches employ whole cells expressing the genes encoding
CYP, Fd, and FdR (Scheme 1A). As an alternative, an H O -
yield. Hydroxylation of VD strictly depends on a cyclodextrin-
3
assisted isomerization of VD into preVD , the actual enzy-
3
3
matic substrate. This facile and robust method developed for
5OHVD synthesis is a novel example for the concept of
2
3
substrate-engineered catalysis and offers an attractive alter-
2
2
native to chemical or O /electron-donor-dependent enzymatic
procedures.
dependent peroxygenase has been used for hydroxylation of
low concentrations of VD and ergocalciferol (VD ; Sche-
me 1B).
2
3
2
[
7]
V
itamin D (VD , cholecalciferol, 1) is generally known for
3 3
its regulatory function in calcium and phosphorous homeo-
stasis, but it is also recognized as an antiproliferative factor in
[1]
dividing cells and tissues. VD is formed from proVD (7-
3
3
dehydrocholesterol, 3) by UV-B irradiation to give the seco
form, followed by a thermal [1,7] sigmatropic hydrogen shift.
It is converted into its biologically active form by consec-
utively acting cytochrome P450 monooxygenases (CYPs):
hydroxylation in the liver yields 25-hydroxy-VD3
(
(
25OHVD , calcidiol, 2), which is converted into calcitriol
3
1a,25(OH) VD ) in the kidney. Insufficient levels of biolog-
2
3
ically active VD are linked to numerous forms of cancer but
3
[
*] M. Sc. M. Warnke, T. Jung, Dr. J. Dermer, K. Hipp, Prof. Dr. M. Boll
Faculty of Biology - Microbiology
Albert-Ludwigs-Universität Freiburg
Scheme 1. Enzymatic conversions of VD (1) and proVD (3) into
3 3
Schänzlestrasse 1, 79104 Freiburg (Germany)
E-mail: matthias.boll@biologie.uni-freiburg.de
25OHVD (2) and 25OHproVD (4), respectively. A) Hydroxylation of
3 3
1 by O -dependent CYPs in the presence of an electron-donor system
2
including NAD(P)H, Fd, and FdR. B) Hydroxylation of 1 by H O -
S. Ferlaino, Dr. A. Fries, Prof. Dr. M. Müller
Institute of Pharmaceutical Sciences
Albert-Ludwigs-Universität Freiburg
2
2
dependent peroxygenase. C,D) Water- and K [Fe(CN) ]-dependent hy-
3
6
droxylation of 1 and 3 by C25DH. (A) and (B) were previously
[6,7]
established, (C) and (D) were studied in this work.
Albertstrasse 25, 79104 Freiburg (Germany)
Dr. N. Jehmlich, Prof. Dr. M. von Bergen
Department of Proteomics
Helmholtz Centre of Environmental Sciences
Permoserstr. 15, 04318 Leipzig (Germany)
A fundamentally different mode of enzymatic steroid
side-chain hydroxylation was identified during the anaerobic
degradation of cholesterol to CO in the denitrifying bacte-
rium Sterolibacterium denitrificans: the regioselective hy-
droxylation of cholesterol into 25-hydroxycholesterol with
Supporting information for this article (including experimental
details) is available on the WWW under http://dx.doi.org/10.1002/
anie.201510331.
2
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[8]
water catalyzed by steroid C25 dehydrogenase (C25DH).
This enzyme belongs to the DMSO reductase (DMSOR)
family of Mo enzymes that catalyze O -independent hydroxy-
2
[9]
and oxo-transfer reactions. A CÀH bond functionalization
with water to a tertiary alcohol via a carbocation transition
[
8]
state has been proposed. C25DH transfers electrons to
electrochemically regenerative electron acceptors such as
K [Fe(CN) ] (Scheme 1C). The genome of S. denitrificans
3
6
contains at least eight homologous genes encoding putative
[
8]
Mo-dependent steroid hydroxylases,
thus making this
organism a promising biological platform for water-depen-
dent hydroxylations of various steroids. In this work, we
explored the use of S. denitrificans enzyme(s) for regioselec-
tive synthesis of the biologically relevant hydroxylated forms
of VD (1) and proVD (3). The hydroxylated 25OHproVD
3
3
3
(
4, Scheme 1D) can easily be converted into 25OHVD (2) by
3
[10]
established UV irradiation methods.
Figure 2. Properties of the enzymatic hydroxylation of 1 and 3.
In the presence of 2-hydroxypropyl-b-cyclodextrin
HPCD), crude extracts from S. denitrificans anaerobically
A) Time-dependent conversion of 1 into 2 in the presence of 0.5 mm
AgNO . B) Time-dependent conversion of 3 into 4. Both reactions
(
3
contained 15% HPCD C) HPCD dependence of the conversion of
grown with cholesterol catalyzed the K [Fe(CN) ]-dependent
3
6
1
1
and 3. D) Substrate concentration dependence of the conversion of
and 3.
conversion of VD (1) and proVD (3) into the single products
3
3
2
and 4 (Figure 2A,B), respectively, with substrate conver-
sions of > 99% (2) and 97% (4; Figure 1A,B). After the
dependent on electron acceptors with standard redox poten-
3
À
tials higher than 200 mV, such as [Fe(CN)6] (ferricyanide),
with the highest initial rates between 5–10 mm K [Fe(CN) ].
3
6
This rather high value can be explained by competing
unspecific K [Fe(CN) ] oxidizing reactions in cell extracts.
3
6
The products formed from 1 and 3 were analyzed by
chemical ionization mass spectrometry. For the product of
+
1
(M = 384.65), compound 2, an ion with m/z [M+H] of
r
4
01.3, was determined, thus indicating the addition of
a hydroxy group (Figure S1 in the Supporting Information).
Likewise, the conversion of 3 (M = 384.65) yielded a product
r
+
with an identical m/z [M+H] (Figure S2). Both results
strongly suggest the hydroxylation of VD (1) and proVD
3
3
(
3).
To unambiguously determine the regioselectivity of the
reaction, the products were characterized by NMR spectros-
1
13
Figure 1. Enzyme assays for the hydroxylation of 1 and 3. A,B) Ultra-
performance liquid chromatography (UPLC) analysis of the enzymatic
conversion of 1 and 3 into their hydroxylated products 2 and 4,
respectively. C,D) UV spectra of 1–4.
copy. The H and C NMR spectra of authentic 1 and 3
standards were highly similar to their products 2 and 4,
respectively, with the exception of the chemical shifts for
1
13
C26,C27 methyl groups ( H spectra), and C25 ( C spectra).
For 1 and 3, the C26 and C27 methyl groups are observed as
1
1
conversion of VD (1) reached 92% (16 h), the product 2 was
doublets at d = 0.86 ppm in the H spectra. For 2 and 4, the H
3
slowly degraded by unspecific enzymatic reactions, which
could be fully prevented by the addition of 0.5 mm AgNO3,
a known inhibitor of the downstream reactions of steroid
catabolism. The UV/Vis spectra of the products were
identical to those of the substrates, which is in agreement
with side-chain hydroxylations distant from the conjugated
triene (1) and diene (3) chromophors (Figure 1C,D).
NMR signal for CH -26,27 is observed as a singlet at d =
3
13
1.23 ppm. A downfield shift is observed in the C NMR
spectrum for the C25 signal (d = 71.1 ppm versus 22.8 ppm for
1 and 3), thus indicating the hydroxylation of C25, as
[8]
[
11]
described for 25-hydroxycholest-4-en-3-one.
1
13
The H and C NMR spectroscopic data are in perfect
agreement with those published for 25OHVD3.
[14]
The
Conversion of 1 and 3 was HPCD dependent, with an
optimum at 15% (w/v; Figure 2C). The initial rate of the
complete NMR spectroscopic data are presented in the
1
13
Supporting Information ( H and C NMR spectra/shifts, 2D
1
reaction followed Michaelis–Menten kinetics, with vmax
=
NMR spectra for 2, and H NMR spectrum of 4; Figures S3–
À1
1
.48 Æ 0.05 nmolmin per mg crude extract protein for VD3
S5 and Table S1).
(1), which is threefold higher than the rate of proVD (3)
To identify the enzyme(s) involved in VD (1) hydroxyl-
3
3
À1
conversion (0.47 Æ 0.02 nmolmin per mg crude extract
ation, we enriched the activity from extracts of cells grown
with cholesterol. After four chromatographic steps, four
protein; Figure 2D). The conversions of 1 and 3 were strictly
1
882
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Figure 3. Enrichment of the VD (1) hydroxylating enzyme. A) Relative
3
specific conversion rates of VD (1, grey columns) and cholest-4-en-3-
3
one (black columns) obtained during the solubilization and purifica-
tion steps. a) cell extract; b) solubilized membrane fraction; c–f) frac-
tions obtained after chromatography on DEAE-Sepharose, ceramic
hydroxyapatite, Reactive Red agarose, and Mono Q anion exchange
chromatography, respectively. B) SDS gel of enriched enzyme after four
chromatographic steps. Left lane: bands 1–3 were identified as abg-
subunits of C25DH; band 4 is a degradation product of band
1
(Table S2). Right lane: Molecular mass standard with masses in kDa
as indicated.
Scheme 2. Proposal for HPCD-dependent hydroxylation of VD by
3
C25DH. In the presence of HPCD, the equilibrium between VD (1)
3
3
and preVD (5), which are interconverted through a thermal [1,7]
hydrogen shift, is shifted to the right. The cZc conformer of 5 is likely
the substrate for C25DH. The hydroxylated 6 was not detected owing
to rapid isomerization to 2.
protein bands were highly enriched with a specific VD (1)
3
À1
À1
hydroxylation activity of 80 nmolmin mg (Figure 3A).
Mass spectrometric analysis of tryptic digests of the enriched
protein bands identified the abg-subunits of C25DH
(
JQ292991-3) plus a fourth band corresponding to a degrada-
tion product of the a-subunit (JQ292991; Table S2). After
each purification step, the relative specific hydroxylation
[8]
rates of VD (1) and cholest-4-en-3-one remained almost
3
constant in all activity-containing fractions (around 70% with
1), thus confirming that both reactions are catalyzed by the
same enzyme (Figure 3B). VD (1) hydroxylation activity was
3
highest in extracts of cells grown with cholesterol (100%),
and significantly lower in cells grown with sitosterol (18%),
ergosterol (15%), or stigmasterol (5%), which is in line with
the induction of C25DH during growth with cholesterol.
The conversion of cholest-4-en-3-one and proVD (3) by
C25DH was observed in the presence of various detergents
3
Figure 4. UPLC analysis of enzymatic VD (1) hydroxylation in the
presence of HPCD after steroid extraction with n-hexane. At t=0 h,
3
and HPCD. In contrast, conversion of VD (1) was negligible
3
before enzyme addition, around 80% of the added VD was HPCD-
3
when HPCD was replaced by a number of commonly used
detergents (Figure S6). This finding suggests that HPCD plays
a specific role in VD3 conversion rather than serving as
dependently isomerized into preVD (5).
3
a general solubilizing agent. The equilibrium preVD (5) Ð
trast, during extraction with ethyl acetate, preVD (5) was
3
3
VD (1) through thermal [1,7] sigmatropic rearrangement lies
never identified during VD (1) hydroxylation (Figure 1A).
3
3
far to the right in aqueous detergent solutions (Scheme 2,
upper reaction). However, in the presence of HPCD, an
To substantiate the HPCD-dependent conversion of VD (1)
3
[
12]
into preVD (5) as an essential process for enzymatic side-
3
inclusion complex is formed at a 2:1 ratio (HPCD/VD ), in
chain hydroxylation, we replaced VD3 (1) by authentic
3
which the thermodynamically unfavorable cZc conformer of
preVD (5) and observed an identical product pattern as
3
[12]
preVD3 (5) is stabilized.
This finding led us to the
during VD (1) conversion (Figure S7). The rates of VD (1)
3
3
assumption that preVD3 (5) rather than VD3 (1) is the
actual substrate of C25DH.
and preVD (5) hydroxylation were nearly identical, thus
3
suggesting that the HPCD-dependent conversion of VD (1)
3
By replacing the commonly used ethyl acetate for steroid
into preVD (5) is not rate limiting.
3
extraction by n-hexane, more than 80% of VD (1, 1 mm)
In summary, the results indicate that preVD (5) but not
3
3
added to the HPCD-containing assay mixture was identified
VD (1) is the substrate for C25DH. The failure to detect any
3
as preVD3 (5) by UPLC analysis. In the course of the
trace of a 25-OH-preVD3 intermediate indicates a rapid
enzymatic reaction, preVD (5) was time-dependently con-
isomerization into 25OHVD (Scheme 2).
3
3
verted into 25OHVD (2), with virtually no formation of the
The procedure presented in this work represents a new
enzymatic route for the regioselective hydroxylation of VD3
3
expected 25OHpreVD (6) intermediate (Figure 4). In con-
3
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deficiencies and to satisfy the increasing demand for mon-
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Acknowledgements
This work was funded by DFG (BO 1565/11-1) in frame of the
Scientific Priority Programme SPP1319. We thank Dr. P.
Bisel, Freiburg, for help with NMR measurements.
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Keywords: biotechnology · CÀH activation · enzyme catalysis ·
2
steroids · vitamins
2
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