CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

Article abstract of DOI:10.1002/cbic.202000735

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

Full text of DOI:10.1002/cbic.202000735

Combining Chemistry and Biology
Accepted Article
Title: CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by
Substrate Modifications and Protein Engineering
Authors: Paula Bracco, Hein J. Wijma, Bastian Nicolai, Jhon Alexander
Rodriguez Buitrago, Thomas Klünemann, Agustina Vila,
Patrick Schrepfer, Wulf Blankenfeldt, Dick B. Janssen, and
Anett Schallmey
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000735
Link to VoR: https://doi.org/10.1002/cbic.202000735
0
1/2020

ChemBioChem
10.1002/cbic.202000735
CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by
Substrate Modifications and Protein Engineering
[
a]
[b]
[a]
[d]
Paula Bracco, Hein J. Wijma, Bastian Nicolai, Jhon Alexander Rodriguez Buitrago,
[
c]
[a]
[d]
[c,d]
Thomas Klünemann, Agustina Vila, Patrick Schrepfer, Wulf Blankenfeldt,
Dick B.
[b]
[d]
Janssen and Anett Schallmey*
a
Biocatalysis, Institute of Biotechnology, RWTH Aachen University, Worringerweg 3,
Aachen, Germany
b
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, Nijenborgh 4, Groningen, Netherlands
c
Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstr. 7,
3
8124 Braunschweig, Germany
d
Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität
Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany
*
Corresponding author:
Prof. Dr. Anett Schallmey
Institute for Biochemistry, Biotechnology and Bioinformatics
Technische Universität Braunschweig
Spielmannstr. 7
3
8106 Braunschweig
Germany
email: a.schallmey@tu-braunschweig.de
Tel.: +49 531 391 55400
Fax: +49 531 391 55401
URL: https://www.tu-braunschweig.de/bbt/biochem
1
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ChemBioChem
10.1002/cbic.202000735
Abstract
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of
steroids with high regio- and stereoselectivity at the 16-position. Using protein engineering
and substrate modifications based on the crystal structure of CYP154C5, an altered
regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion
of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-
hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD
simulation, this altered regioselectivity appeared to result from an alternate binding mode of
the steroid in the active site of mutant F92A. MD simulation further suggested that water
entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-
hydroxylation was observed for wild-type CYP154C5 in the conversion of 5-androstan-3-
one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the
structure-function relationship of this cytochrome P450 monooxygenase for steroid
hydroxylation.
2
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ChemBioChem
10.1002/cbic.202000735
Introduction
Cytochrome P450 monooxygenases (P450s or CYPs) are hemoproteins carrying a
[
1]
heme b molecule covalently linked to a cysteine side chain. From a biocatalytic perspective,
they are remarkable enzymes as they are able to catalyze the selective hydroxylation of non-
activated carbon atoms using molecular oxygen.[2,3] For the activation of molecular oxygen
during the catalytic cycle, they require electrons  coming from NAD(P)H  which are in
[
4]
most cases delivered to the monooxygenase by additional redox partners. One of the most
important industrial uses of these cytochrome P450 monooxygenases is their application in
steroid synthesis in the pharmaceutical industry due to their remarkable selectivity in steroid
hydroxylation. Well-known examples include the 11-hydroxylation of 11-deoxycortisol
(Reichstein S) to hydrocortisone by Curvularia sp. or the conversion of progesterone to
cortisone by Rhizopus sp.[5–8] Though P450s are already applied on industrial scale, there is
always the need for yield improvement, including the increase in hydroxylation specificity
(e.g. fewer by-products), the need for an altered selectivity of the enzyme to hydroxylate, e.g.
[
9]
also new sites in a known substrate or the adaptation of a known P450 to a new substrate. In
that respect, protein engineering has proven to be a powerful tool to alter enzyme
characteristics such as activity and selectivity. One example, reported by Kille et al., is the
generation of P450BM3 mutants hydroxylating testosterone selectively either at 2β- or 15β-
position, while the starting mutant, P450BM3 F87A, forms a 1:1 mixture of 2β- and 15β-
hydroxytestosterone.[10] In this case, mutants were generated by iterative combinatorial active-
site saturation mutagenesis, a protein engineering approach that is significantly facilitated by
the availability of structural information for a given protein.
Recently, the crystal structure of CYP154C5, a cytochrome P450 monooxygenase
from Nocardia farcinica, was elucidated in the presence of four steroid substrates.[11] This
enzyme catalyzes the highly regio- and stereoselective hydroxylation of different pregnans
and androstans producing exclusively the corresponding 16α-hydroxylated products.[12] As the
natural redox partners of this P450 monooxygenase are unknown, putidaredoxin (Pdx) and
putidaredoxin reductase (PdR) from Pseudomonas putida can be applied in bioconversions to
supply CYP154C5 with electrons. The active site pocket of CYP154C5 forms a hydrophobic
“tube” with two opposite polar regions at both ends. These polar regions are occupied by
glutamine 239 and glutamine 398 forming hydrogen bond interactions with the hydroxyl or
ketone functionalities of steroids at positions C3 (via water molecules) and C17.[11]
Additionally, several hydrophobic interactions between enzyme and steroid substrate within
3
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ChemBioChem
10.1002/cbic.202000735
the active site could be identified. With the help of the crystal structure the remarkably high
regio- and stereoselectivity of CYP154C5 towards steroids 1-6 (Figure 1) was explained.[11]
With the future goal to modify the enzyme’s regioselectivity in steroid hydroxylation
reactions, we herein aimed to explore the selectivity of CYP154C5 in more detail based on
the previously obtained structural insight. To this end, selected active-site residues of
CYP154C5, mediating important enzyme-substrate interactions, were mutated by site-directed
mutagenesis, and resulting mutants were applied in bioconversions with steroids 1-6. In a
complementary approach, steroid substrates lacking oxyfunctional groups at C3 and C17,
which otherwise enable hydrogen bonding with active site residues of CYP154C5, were tested
in bioconversions with the wild-type enzyme.
Figure 1. Chemical structures of previously tested (1-6)[11,12] as well as five new steroid substrates (7-
11) used in bioconversions with CYP154C5 (wild type and mutants), Pdx and PdR in this study.
4
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ChemBioChem
10.1002/cbic.202000735
Results and Discussion
CYP154C5 mutagenesis
Based on the CYP154C5 crystal structure and a detailed analysis of the enzyme active
site in the presence of different steroid substrates,[11] four active-site residues have been
identified to play an important role in steroid binding. Among them are the two glutamines at
positions 239 and 398 forming hydrogen bonds with the carbonyl or hydroxyl groups at C3
(via a water molecule) and C17 of the steroid substrates (Figure 2). Moreover, residues M84
and F92 are involved in hydrophobic interactions with the steroid backbone. Specifically,
M84 interacts with C11 and C12 of ring C as well as methyl substituents (C18 and C19) on
the steroid backbone, while residue F92 forms hydrophobic contacts with C5, C6, C7, and C8
of ring B.[11] Thus, residues M84, F92, Q239 and Q398 were selected for mutagenesis to
investigate their impact on steroid binding and CYP154C5´s selectivity in steroid
conversions. To this end, single alanine mutations were prepared using site directed
mutagenesis. Afterwards, wild-type CYP154C5 as well as mutants M84A, F92A, Q239A and
Q398A were heterologously produced in E. coli C43 (DE3) and purified by anion exchange
and affinity chromatography (Figure S1). Similarly, redox partners Pdx and PdR from P.
putida, which are required for P450 activity, were produced in E. coli C43 (DE3) and
subsequently purified by anion exchange and hydrophobic interaction chromatography
(Figure S1).
5
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ChemBioChem
10.1002/cbic.202000735
Figure 2. Superposition of the active sites of the four CYP154C5-steroid complexes (PDB codes:
4J6B, 4J6C, 4J6D and 4JBT) with the four amino acid residues M84, F92, Q239 and Q398 involved in
steroid binding.
Steroid conversion by CYP154C5 mutants
In order to analyze the influence of the different mutations on steroid binding and
catalysis by CYP154C5, dissociation constants (K ), turnover numbers (TONs) and coupling
D
efficiencies of the mutants towards steroids 1-6 were determined. In the absence of substrate,
P450 enzymes exhibit an absorbance maximum around 420 nm. Upon substrate binding, a
water molecule is removed as the sixth ligand at the heme iron.[13] This causes a spin shift of
the heme iron which, in type I spectral changes, involves a shift of the P450 absorbance
maximum to approximately 390 nm. From the peak-to-trough difference in absorbance (A)
between P450 with high spin iron (A390) and low spin iron (A420) dissociation constants can be
inferred. Using the four different CYP154C5 mutants and steroids 1-6, K values for almost
D
all combinations could be determined (Table 1; respective plots are displayed in Figures S2-
S6). Some mutants, however, yielded only a partial or no spectral shift upon substrate addition
(
Figure S7), even at high steroid concentration, hampering K determination. A missing
D
spectral shift upon substrate addition could indicate that the respective steroid does not bind
anymore in the mutated active site of CYP154C5. It is also possible, however, that due to the
mutation in the active site, the bound substrate is not positioned anymore close to the heme
iron to displace the water molecule as the sixth ligand.
Table 1. Dissociation constants (K
values were determined at 30°C for the His-tagged CYP154C5 mutants using the quadratic tight-
binding equation.[14] All values are the result of duplicate measurements given as mean ± SD.
(nM)
D
) of CYP154C5 wild type and mutants for steroid substrates 1-6.
K
D
K
D
CYP154C5
M84A
CYP154C5
F92A
CYP154C5
Q239A
CYP154C5
Q398A
Substrate
CYP154C5
[
a]
-/+ [b]
Pregnenolone (1)
62 ± 14

<1
<1
<1
<1
<1
65 ± 21
687 ± 43
34 ± 21
Dehydroepi-
androsterone (2)
<
1
-/+ [b]
-/+ [b]
1017 ± 32
209 ± 12
512 ± 17
1481 ± 33
Progesterone (3)
15 ± 10
2 ± 7
18 ± 10
Androstene-
dione (4)
1
3130 ± 234
5710 ± 161
572 ± 151
747 ± 45
Testosterone (5)
6
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ChemBioChem
10.1002/cbic.202000735
[a]
-/+ [b]
[a]
Nandrolone (6)
100 ± 17

13 ± 6

[
[
a]
b]
No shift to high-spin heme observed.
Only partial shift to high-spin heme observed.
Notably, obtained K
wild type CYP154C5, indicating a substantial influence of the mutated active-site residues on
steroid binding. The main exception is CYP154C5 Q239A for which the obtained K values
were even lower than for wild-type CYP154C5. This suggests a positive influence of
D
values of the alanine mutants were in many cases higher compared to
D
mutation Q239A on steroid binding. The exact molecular reasons, however, are difficult to
anticipate, as the exchange of glutamine by alanine at this position will actually result in a loss
of a hydrogen bond, via a water molecule, to the oxygen atom at C3 of the steroids. Here, a
crystal structure of CYP154C5 Q239A with one of the steroids 1-6 bound in the active site
could be solved in the future to gain deeper insight. Furthermore, binding of pregnenolone (1)
and progesterone (3) to CYP154C5 was hardly affected by amino acid exchange Q398A,
whereas significantly higher K values were obtained for all other steroid substrates. Removal
D
of the glutamine side chain in CYP154C5 Q398A leads to a loss of a hydrogen bond to the
C17-substituent oxygen of the steroid substrate, and generates space that could result in a
possible movement of steroids 2, 4, 5 and 6 in the active site. In contrast, steroids 1 and 3
carry a more spacious acetyl side chain at C17, which could fill this space resulting in a
tighter binding. Interestingly, mutation M84A in CYP154C5 affected substrate binding the
most among all tested variants. More than 100-fold lower binding affinities compared to wild-
type CYP154C5 were obtained with androstenedione (4) and testosterone (5). Additionally,
no spectral shifts of CYP154C5 M84A were observed with steroids 1 and 6, while substrates
2
and 3 induced only partial shifts. This clearly emphasizes the importance of position M84
for steroid binding and correct positioning within the active site of CYP154C5.
Turnover numbers and coupling efficiencies of the purified mutants were determined
in the conversion of steroids 1-6, together with Pdx and PdR as redox partners (Tables 2 and
3
). Mutants CYP154C5 M84A, CYP154C5 F92A and CYP154C5 Q398A exhibited
significantly decreased TONs in comparison to the wild-type enzyme, independent of the used
substrate, while for mutation Q239A the TONs were less affected (Table 2). This is in general
agreement with the observed higher K values obtained for mutants CYP154C5 M84A,
D
CYP154C5 F92A and CYP154C5 Q398A. Interestingly, CYP154C5 M84A converted
pregnenolone (1) to a small extent even though no shift from low to high spin could be
detected. Such a behavior was previously observed in the case of cytochrome P450 from
7
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ChemBioChem
10.1002/cbic.202000735
Bacillus megaterium, CYP106A2, where no change in absorbance was obtained upon addition
of deoxycortisone (DOC) although this substrate is converted by this enzyme producing 15β-
hydroxydeoxycortisone.[15] Simgen et al., however, proved by FT infrared spectroscopy using
the stretch vibration of the heme iron CO-ligand that DOC indeed enters the active site and
binds close to the heme prosthetic group.[15]
Similarly, obtained coupling efficiencies of the CYP154C5 mutants in the conversion
of steroids 1-6 were, in many cases, also negatively affected (Table 3). This is especially
evident for substrates pregnenolone (1) and progesterone (3) as well as for coupling
efficiencies of mutant CYP154C5 M84A with all tested steroids. In contrast, coupling
efficiencies of CYP154C5 F92A with nandrolone (6), CYP154C5 Q239A with 3, 5 and 6 as
well as coupling efficiencies of CYP154C5 Q398A with testosterone (5) are still similar to the
wild-type values.
Table 2. Turnover numbers (TON) of CYP154C5 mutants in the conversion of steroids 1-6. TON
were determined at 30°C based on the highest observed substrate consumption rate in each reaction.
Measurements were performed in duplicate. Values are given as mean ± SD.
TON (µmol substrate consumed µmolP450 min-1)
-1
CYP154C5
M84A
CYP154C5
F92A
CYP154C5
Q239A
CYP154C5
Q398A
Substrate
CYP154C5[a]
Pregnenolone (1) 4.47 ± 0.36
0.20 ± 0.00
0.75 ± 0.09
2.52 ± 0.12
1.53 ± 0.14
0.70 ± 0.05
0.85 ± 0.16
1.48 ± 0.26
1.02 ± 0.07
1.68 ± 0.07
1.75 ± 0.12
1.33 ± 0.09
2.48 ± 0.02
2.07 ± 0.24
4.33 ± 0.05
2.42 ± 0.05
1.32 ± 0.12
1.28 ± 0.02
0.93 ± 0.14
1.22 ± 0.21
1.45 ± 0.07
1.77 ± 0.19
0.57 ± 0.09
Dehydroepi-
androsterone (2)
5
.1 ± 0.11
Progesterone (3)
5.7 ± 0.22
Androstene-
dione (4)
3
.14 ± 0.36
1.77 ± 0.06
1.33 ± 0.10
Testosterone (5)
[b]
[b]
Nandrolone (6)


[
[
a]
Data taken from [11] for comparison
No conversion observed
b]
8
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10.1002/cbic.202000735
Table 3. Coupling efficiencies of CYP154C5 mutants in the conversion of steroids 1-6. Measurements
were performed in duplicate. Values are given as mean ± SD.
Coupling efficiency (%)
CYP154C5
M84A
CYP154C5
F92A
CYP154C5
Q239A
CYP154C5
Q398A
Substrate
CYP154C5[a]
Pregnenolone (1)
68 ± 1
34 ± 6
60 ± 7
34 ± 1
23 ± 14
39 ± 4
40 ± 8
55 ± 0
22 ± 9
64 ± 3
46 ± 3
52 ± 18
27 ± 5
90 ± 4
38 ± 8
66 ± 6
64 ± 8
34 ± 3
53 ± 2
41 ± 6
46 ± 0
51 ± 7
68 ± 1
Dehydroepi-
androsterone (2)
8
6 ± 5
84 ± 3
3 ± 1
Progesterone (3)
Androstene-
dione (4)
8
Testosterone (5)
62 ± 5
40 ± 7
[b]
[b]
Nandrolone (6)


[
[
a]
Data taken from [11] for comparison
No conversion observed
b]
In almost all cases, the CYP154C5 mutants still formed the corresponding 16α-
hydroxylated products in steroid conversions of 1-6. Hence, regioselectivity of the single
mutants was generally not affected, except for the conversion of progesterone (3) by mutant
CYP154C5 F92A. Here, formation of a second hydroxylation product was observed (Figure
S12), which was identified as 11-deoxycorticosterone (hydroxylation at position 21) by NMR
analysis (Scheme 1). Both products, 16α- and 21-hydroxylated progesterone, were produced
in a ratio of 4:1 by CYP154C5 F92A. Hydroxylation of progesterone as well as 17α-
hydroxyprogesterone at C21 yielding 11-deoxycorticosterone and 11-deoxycortisol,
respectively, are important steps in adrenal steroidogenesis required for the synthesis of
glucocorticoids and mineralocorticoids. In human, CYP21A2 is the responsible enzyme
catalyzing this step. Interestingly, for CYP21A2 the formation of 16α-hydroxyprogesterone as
side product has been reported as well.[16] Moreover, exchange of valine 359 by alanine
yielded a mutant with significantly increased hydroxylation in 16α position resulting in a ratio
of 21-hydroxyprogesterone to 16α-hydroxyprogesterone of 60:40, while mutant CYP21A2
V359G gave even 90% 16α-hydroxyprogesterone.[17]
To reveal the structural basis of the change in regioselectivity of mutant CYP154C5
F92A in progesterone hydroxylation, this enzyme-substrate complex was studied by
computational tools.
9
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ChemBioChem
10.1002/cbic.202000735
Scheme 1. Conversion of progesterone (3) by CYP154C5 wild type and mutants M84A,
F92A, Q239A and Q398A together with redox partners Pdx and PdR yielding 16α-
hydroxyprogesterone. Only in case of CYP154C5 F92A a second hydroxylation product,
carrying a hydroxyl group at C21, was observed.
Modeling of CYP154C5 F92A
A structural model of the compound I intermediate of the F92A mutant was generated and
used in docking and molecular dynamics simulations. As a reference, a crystal structure of
wild-type CYP154C5 with progesterone (3) bound was converted to the compound I state
(
Figure 3-A) and subjected to docking as well.[11] The docking simulations with the F92A
mutant suggested two possible binding orientations for substrate 3. One orientation is similar
to the one found in the crystal structure (Figure 3-C), whereas the second is clearly different:
progesterone is reoriented with its A and B ring now occupying the space created by the F92A
mutation and only the hydrogens of carbon 21 are close to the reactive oxygen of compound I
(Figure 3-B). This alternative orientation of the substrate would cause extreme steric
hindrance, if F92 would remain in the position that is observed in all X-ray structures; the
aromatic ring of F92 would have to interlock with the A or B ring of the steroid.
1
0
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10.1002/cbic.202000735
Figure 3. The modeled orientations of progesterone (3) can explain selective oxidations at the 21 and
6α positions. For each structure, 10 MD snapshots are shown. Carbon 16 and 21 of the progesterone
3) substrate are indicated with a black-white checker pattern. A) Wild type CYP154C5, where both
1
(
the hydrogens at the 21 and 16 position are close to the compound I oxygen, suggesting the result
will be selective oxidation of the more reactive secondary carbon atom 16 instead of the primary
carbon atom 21; B) F92A with progesterone (3) bound in an alternative orientation, in which only
oxidation at the 21 position is possible. C) F92A is also predicted to occur with progesterone (3)
bound in a wild type-like orientation, enabling 16 oxidation.
The first step in P450-catalyzed hydroxylation is the abstraction of a hydrogen atom from the
substrate by the electrophilic oxygen of the compound I intermediate.[18] The predicted
reactivity of different enzyme-substrate complexes was therefore examined by molecular
dynamics (MD) simulations with scoring of near-attack conformations (NACs) in which
substrate hydrogens approach the oxygen of the compound I intermediate (Figure S8). For
each complex, three independent 22 ns MD simulations were performed. The enzyme-
substrate complexes were stable (Figure 4) and the simulations gave reproducible results. In
simulations of progesterone (3) in complex with wild-type CYP154C5, both the 16α and 21
hydrogen atoms stayed close to the reactive oxygen atom (Figure 4-A). The overall X-ray
structure was maintained and high percentages of NACs were found for both positions (Table
4
). The observation that only 16α hydroxylation took place with the wild-type enzyme is in
agreement with the higher reactivity of this secondary carbon atom compared to the primary
carbon atom at position 21,[19] even though the latter might be influenced by the flanking
carbonyl group.
1
1
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ChemBioChem
10.1002/cbic.202000735
With the substrate oriented in the F92A mutant like it is in the wild-type enzyme, the
distances and NAC percentages for the 16α and 21 hydrogens calculated from the simulations
were similar to those found with the wild-type CYP154C5 (Figure 4-C). In contrast, MD
simulations of the F92A mutant with progesterone (3) bound in the alternative orientation
(Figure 4-B) showed that in this case only the 21 position can undergo oxidation. The distance
between the 16α hydrogen and the reactive oxygen is predicted to exceed 6 Å, making
hydrogen abstraction impossible and only the 21 hydrogens gave significant levels of NACs
(Table 4). Thus, the modeling suggests that the F92A mutation provides an additional
progesterone (3) binding mode that is particularly suitable for oxidation at the 21 position.
Also Pallan et al. suggested that two alternate binding modes of 3 in CYP21A2’s active site
are responsible for the formation of 21- and 16α-hydroxylated products by this enzyme based
on an observed partial burst in pre-steady state kinetics.[20] Furthermore, two alternative
orientations for progesterone (3) binding have also been observed for CYP260A1 by
molecular docking, explaining the low selectivity of this CYP in progesterone conversion.
Moreover, through targeted mutagenesis of active-site residue S276, one or the other binding
orientation could be suppressed yielding two highly regio- and stereoselective CYP260A1
mutants, which formed either 1α- or 17α-hydroxyprogesterone selectively.[21]
A
B
C
16a
21
0
5 10
20
0
5 10
20
0
5 10
20
time (ns)
Figure 4. Distance of the substrate‘s (3) hydrogen atoms to the oxygen of compound I. Because there
are three identical 21 hydrogen atoms per substrate, only the shortest distance is shown at each time-
point. Also for clarity, for each modeled enzyme substrate complex only the first out of the three
independent MD simulations is shown. A) Wild-type CYP154C5 with progesterone (3) bound. B)
CYP154C5 F92A with progesterone (3) bound in the alternative orientation; C) CYP154C5 F92A
with progesterone (3) bound in the native-like orientation.
1
2
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10.1002/cbic.202000735
Table 4. Near attack conformations of wild-type (WT) CYP154C5 and mutant F92A with
progesterone as ligand. Potential oxidation sites of progesterone are given.
Structure simulated by molecular dynamics
NACs during MD simulation (%)
16 oxidation
21 oxidation
WT + progesterone
22
0
7
42
5
F92A + progesterone in alternative orientation[a]
F92A + progesterone in native like orientation[b]
2
[
[
a]
b]
Also 0.4% NACs were observed for 12 oxidation.
Additionally, 0.3% NACs for oxidation at position 17 was recorded.
Furthermore, CYP154C5 F92A with progesterone (3) also gave the highest
uncoupling (Table 3). Uncoupling is commonly observed with P450 mutants and is a highly
relevant challenge as it hinders their use in applied catalysis.[22] We found that substrate 3 is
more mobile in the active site of mutant F92A. The root mean square fluctuations (RMSF) of
this substrate are higher in the mutant than in wild type CYP154C5 (Figure S9). This suggests
that the shape-complementarity between enzyme and substrate is not optimal in the mutant.
Also, while in case of the wild type the closest water stayed at a 7 Å distance from the
reactive oxygen during the entire MD simulations (in agreement with a good enzyme-
substrate shape complementarity), with mutant F92A a water molecule did approach the
reactive oxygen for both substrate orientations (Figure 5). Some of the observed distances are
less than 2 Å. Moreover, the intruding water forms a H-bond with the reactive oxygen. While
the encroaching water does not drive out the substrate (Figure 4 as well as Figures S10 and
S11), the H-bonding should change the reactivity of compound I. Uncoupling at the stage of
compound I (the oxidase shunt) involves the addition of two protons and two electrons after
which the reactive oxygen dissipates to water. Mechanistically, it seems unlikely that the
water molecule would protonate compound I (at least not before the latter has been further
reduced by one or two electrons), as compound I already has a strongly positive charge. It
seems more feasible that the H-bonding would decrease the reactivity of compound I or
increase its redox potential, which in either case would increase the chance that compound I
becomes reduced before the substrate had the time to react. Theoretical modeling found that
H-bonding to the sulfur that coordinates the iron has a strong effect on reactivity.[23,24] H-
bonding by water to the reactive oxygen atom of compound I is expected to have even
stronger effects, as it is extremely close to the site of the reaction.
1
3
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ChemBioChem
10.1002/cbic.202000735
A
0
0
0
5
5
5
10
15
15
15
20
20
20
time (ns)
B
10
time (ns)
C
10
time (ns)
Figure 5. Distance between the reactive oxygen atom of compound I and the nearest water molecule
during MD simulation. The results from three independent MD simulations are shown with a black
nd
continuous line (first simulation), with blue long dashes (2 simulation), and with gray short dashes
(
3rd simulation). A) Wild type CYP154C5 with progesterone (3) bound. B) CYP154C5 F92A with
substrate 3 bound in the alternative orientation; C) CYP154C5 F92A with substrate 3 bound in the
native like orientation.
1
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10.1002/cbic.202000735
Bioconversions of new steroid substrates by wild-type CYP154C5
Instead of introducing mutations, an alternative approach to study the enzyme’s
selectivity is the selection of steroid substrates that are either lacking key functional groups
for the enzyme-substrate interaction or that are carrying new features. Hence, based on the
known CYP154C5-steroid complex structures, five new steroid substrates were selected. As
previously reported, oxyfunctional groups at C3 and C17 were shown to form hydrogen bond
interactions with residues Q239 (via a water molecule) and Q398, respectively.[11] Therefore,
steroid substrates lacking one (10 and 11) or both (9) oxyfunctional groups, as well as steroids
containing a larger side chain at position C17 (7 and 8) were chosen to be tested in
bioconversions with wild-type CYP154C5 (Figure 1).
Initially, small-scale reactions employing whole cells or cell-free extract of E. coli
containing wild-type CYP154C5, Pdx and PdR were carried out in order to investigate if
compounds 7-11 are converted. Reactions employing whole cells or cell free extract of E. coli
containing only Pdx and PdR were used as negative controls under the same reaction
conditions. No conversion of finasteride (7) and etiadienic acid ethyl ester (8) by CYP154C5
could be observed, neither employing whole cells nor cell-free extract. This is probably the
result of the larger side-chain at C17 preventing binding of the steroids in the enzyme’s active
site. This is further supported by the fact that 7 and 8 did not induce any spectral shift during
D
K measurements (data not shown). Similarly, CYP154C3 from Streptomyces griseus, a
homologue of CYP154C5 hydroxylating steroids selectively at 16α position as well, is also
unable to convert steroids with bulky substituents at the D ring.[25] In case of ethioallocholane
(9) conversion by CYP154C5, a possible product peak was identified by GC-MS (Figure S15)
though conversion was too low for product isolation. A NIST-library search suggested a
steroid-related structure for this product. Additionally, steroid 9 induced a partial spectral shift
of CYP154C5 during K
D
measurements with a high-spin species content of roughly 50%
(Figure S7-G). These results suggest that ethioallocholane (9) is indeed converted by
CYP154C5 but further tests will be necessary to identify the formed product. In contrast,
conversion of 3-deoxydehydroepiandrosterone (10) led to the formation of several products
(Figure S16), indicating that the regio- and/or stereoselectivity of CYP154C5 was altered.
Moreover, conversion of 5α-androstan-3-one (11) by CYP154C5 resulted in one hydroxylated
product (Figure S20).
In order to elucidate the structures of formed products, whole-cell bioconversions were
performed on preparative scale for substrates 3-deoxydehydroepiandrosterone (10) and 5-
androstan-3-one (11). Similar to the results obtained on analytical scale, several products were
1
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ChemBioChem
10.1002/cbic.202000735
formed in the preparative-scale conversion of 10 by wild-type CYP154C5. Of these products,
two were obtained in sufficient amount and purity for subsequent NMR analysis (see
supplementary information for respective NMR spectra). Results revealed that 16-hydroxy-
3
-deoxydehydroepiandrostendione was formed as the main product (Scheme 2). Interestingly,
the second purified product seems to be the result of a double hydroxylation as indicated by
GC-MS and NMR data (Figures S18 and S19). The exact hydroxylation positions, however,
could not be determined due to low product quantities. Preliminary docking studies with 3-
deoxydehydroepiandrosterone (10) and the compound I model of wild-type CYP154C5
suggest position 4β as potential hydroxylation site in addition to the observed 16α-
hydroxylation (Figure S12-A). Similarly, also product 16α-hydroxy-3-
deoxydehydroepiandrosterone was docked in the active site with position 4β as potential
hydroxylation site (Figure S12-B), which would ultimately result in a double hydroxylation of
1
0. Furthermore, position 2α was identified as potential additional hydroxylation site when
using 4β-hydroxylated 10 as docking substrate (Figure S12-C). Even though these docking
poses were only obtained in silico, they can give a first indication for potential hydroxylation
sites of the other observed products.
Scheme 2. Conversion of 3-deoxydehydroepiandrosterone (10) and 5-androstan-3-one (11)
by CYP154C5 wild type together with redox partners Pdx and PdR and the obtained
hydroxylation products as revealed by NMR.
In contrast, the product formed in the preparative-scale conversion of 5-androstan-3-one
(11) by CYP154C5 was identified as 15-hydroxy-5-androstan-3-one by NMR (Figure S21
and Scheme 2; details on the assignment of the hydroxylation product are given in the
supplementary). This result indicates a change in CYP154C5’s regioselectivity in the
1
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ChemBioChem
10.1002/cbic.202000735
conversion of 11, likely caused by the lack of the functional group at position C17 and/or the
saturated A-ring of the steroid substrate. To gain structural insight into the altered
regioselectivity of wild-type CYP154C5 in the conversion of 11, the P450 was co-crystallized
with this steroid. The overall structure of CYP154C5 is very similar to the previously
determined CYP154C5 structures with C -RMSD values of 0.20-0.37. The electron density of
α
the ligand and the derived model indicate that 5-androstan-3-one (11) binds in a similar
position as androstenedione (4) and testosterone (5) (Figure 6), the latter being hydroxylated
in 16α position by wild-type CYP154C5. Interestingly, however, C15 of steroids 4, 5 and 11
in the corresponding crystal structures of CYP154C5 are in a similar position as C16 of
pregnenolone (1) and progesterone (3) (Figure 6 A), which are also hydroxylated in 16α
position by wild-type CYP154C5. Hence, the crystal structure alone cannot explain the
observed hydroxylation of 5-androstan-3-one (11) in 15α-position. Therefore, MD
simulations (10 trajectories of each 22 ns) of the compound I state of the CYP154C5 structure
with 11 bound in the active site have been performed with scoring of NACs as mentioned
before (see Figure S13). This way, predicted NAC percentages of 0.31% for C15 and 15.8%
for C16 were obtained. This confirms that C15 can be reached in productive conformations by
the oxygen of compound I, even though the probability for attack of C16 seems to be higher
as judged by distance. The question, why only 15-hydroxy-5-androstan-3-one is observed
as product in conversions of 11 by CYP154C5, cannot finally be solved but might be caused
by a higher reactivity of C15 compared to C16. Here, quantum mechanical calculations could
be performed in the future to reveal further insight.
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ChemBioChem
10.1002/cbic.202000735
Figure 6. (a) Superimposition of the crystal structures of CYP154C with bound compound 11
green) and the published steroid complexes [PDB-codes: 4J6B (1, yellow), 4J6C (3, cyan),
J6D (5, pink) and 4JBT (4, magenta)]. (b) Mesh representing a 2F electron density
contoured at a σ level of one carved around the steroids which are colored according to (a).
(
4
F
o c
Additionally, K values, TONs and coupling efficiencies for wild-type CYP154C5 in
D
the conversion of 3-deoxydehydroepiandrosterone (10) and 5-androstan-3-one (11) were
determined. CYP154C5 exhibited a rather high affinity towards substrates 10 and 11 with K
values of 94 ± 52 and 20 ± 16 nM, respectively. In contrast, coupling efficiencies are
D
dramatically decreased in both cases resulting in only 7 ± 6 and 26 ± 10% for steroids 10 and
-1
1
1, respectively. Similarly, obtained TONs (0.67 ± 0.17 and 0.77 ± 0.17 min for 10 and 11,
respectively) are also rather low compared to steroids 1-6. This suggests that steroid
conversion by CYP154C5 is indeed significantly affected if one of the oxyfunctional groups
at C3 or C17 of the steroid backbone, and hence the corresponding hydrogen bond, is missing.
On the other hand, TONs of wild-type CYP154C5 for conversion of 10 and 11 were
determined for the whole-cell system and not isolated enzyme. Hence, the resulting data is not
directly comparable to TONs determined in the conversion of steroids 1-6.
Conclusion
With our study, we were able to demonstrate experimentally that the previously reported high
regioselectivity of CYP154C5 is dependent on the presence of oxyfunctional groups at C3 and
C17 of the steroid substrate, as products with new hydroxylation sites were obtained in
reactions with steroids lacking one of those substituents. Here, hydroxylation of 5-
androstan-3-one in 15α position is especially interesting, as 15α hydroxylation of steroids by
bacterial cytochrome P450 monooxygenases has only been reported once so far, in the
conversion of testosterone and dehydroepiandrosterone by specific mutants of CYP102A1
from Bacillus megaterium.[26] In contrast, the replacement by alanine of active site residues
Q239 and Q398 in CYP154C5, which have been shown to interact with the oxyfunctional
groups through hydrogen bonding, did not alter the enzyme’s regioselectivity but had a
negative impact on catalytic efficiency. Similarly, mutagenesis of residues M84 and F92,
which form hydrophobic interactions with the steroid backbone, resulted in reduced turnover
numbers and coupling efficiency as well as higher dissociation constants for most steroid
substrates tested. This was especially evident for mutant M84A, which confirms the
importance of residue M84 for delimiting the active site pocket and confining the steroid in a
1
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ChemBioChem
10.1002/cbic.202000735
catalytically active position. Moreover, mutation F92A appeared to enable a second binding
orientation of progesterone in the enzyme active site resulting in C21 hydroxylation. Overall,
our data demonstrate the feasibility for future modification of CYP154C5 regioselectivity by
protein engineering and give valuable insight into the structure-function relationship of this
cytochrome P450 monooxygenase for steroid hydroxylation.
Experimental Section
Substrates and chemicals. Pregnenolone (1), progesterone (3), testosterone (5) and
cholesterol were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Dehydroepiandrosterone (2), androstenedione (4), nandrolone (6), finasteride (7), etiadienic
acid ethyl ester (8), ethioallocholane (9), 3-deoxydehydroepiandrostendione (10) and 5α-
androstan-3-one (11) were purchased from Steraloids Inc. (Newport, USA). Cytochrome c
from horse heart, formate dehydrogenase from Candida boidinii, superoxide dismutase,
catalase from bovine liver and hydroxypropyl β-cyclodextrine were purchased from Sigma
Aldrich. All solvents and other chemicals required for the experiments were obtained from
Roth (Karlsruhe, Germany) or Sigma-Aldrich and used without further purification.
Bacterial strains and plasmids. Escherichia coli DH5α (Invitrogen, Carlsbad, CA, USA)
was used for genetic manipulations, while E. coli C43 (DE3) (Lucigen, Middleton, WI, USA)
was used for recombinant gene expressions. Plasmid pET28a(+) was purchased from
Novagen (EMD Biosciences, San Diego, CA, USA). Preparation of plasmid pACYCcamAB
for coexpression of putidaredoxin reductase (CamA or PdR) and putidaredoxin (CamB or
Pdx) from Pseudomonas putida was described elsewhere.[27] Preparation of plasmid
pIT2cyp154c5 was described elsewhere.[12] The gene of CYP154C5 was subcloned from
vector pIT2cyp154c5 into pET28a(+) using restriction sites NdeI and HindIII. The resulting
plasmid was named pET28cyp154C5. Expression of CYP154C5 from vector
pET28cyp154C5 results in a fusion protein with N-terminal His-tag.
Generation of CYP154C5 mutants. Mutants were prepared by QuikChange® site-directed
mutagenesis using the Pfu-Turbo Hotstart PCR Master Mix (Agilent, Waldbronn, Germany)
according to the manufacturer’s instructions. The primers applied in the PCR reactions are
listed in Table S1 in the supplementary. Correct introduction of the mutations was confirmed
by sequencing at GATC Biotech (Konstanz, Germany) and final plasmids were transformed
into E. coli C43 (DE3) for protein expression.
1
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ChemBioChem
10.1002/cbic.202000735
Expression and purification of enzymes. Production of CYP154C5 wild type and its
mutants using E. coli C43 (DE3), as well as expression of camA (PdR gene) and camB (Pdx
gene) using E. coli C43 (DE3) (pACYCcamA) and E. coli C43 (DE3) (pACYCcamB),
respectively, were performed as described elsewhere.[11] Protocols for the production of E.
coli C43 (DE3) (pIT2cyp154C5) (pACYCcamAB) and E. coli C43 (DE3) (pETcyp154C5-
F92A) (pACYCcamAB) whole-cell biocatalysts were also previously described.[12]
Purification of wild-type CYP154C5 and its mutants via N-terminal His-tag, as well as
purification of PdR and Pdx by anion exchange and hydrophobic interaction chromatography
were also performed as previously described.[11]
Enzyme assays. The P450 concentration was measured using CO-difference spectra.[28] The
activity of the purified electron transfer components (ETC) Pdx and PdR was determined by
cytochrome c reduction assay, monitoring the increase in absorbance at 550 nm of reduced
-1
-1
cytochrome c (ε450 = 19.1 mM cm ) in a mixture containing a ratio of PdR:Pdx equal to
:16.[29] Additionally, all enzymes assays were also measured with cell lysate of the respective
3
E. coli C43 (DE3) cells containing P450, PdR and Pdx before whole-cell catalysis as
described elsewhere.[11] Total protein concentration was determined by Bradford assay.[30]
Substrate binding studies. Dissociation constants (K ) of CYP154C5 wild type and
D
CYP154C5 mutants for the different steroids were determined by spectroscopic
measurements upon titration of purified P450 with increasing steroid concentrations.[13] Thus,
purified CYP154C5 carrying an N-terminal His-tag was diluted with 50 mM potassium
phosphate buffer pH 7.4 in order to reach 3 µM final enzyme concentration. To this mixture,
substrate was added in concentrations from 0 to 150 µM. Therefore, three different substrate
stock solutions of 0.1, 0.5 and 1 mM in 0.1-4.5% (w/v) hydroxypropyl-β-cyclodextrin in
deionized water (diH
measured on a Cary 50 spectrophotometer (Agilent, Waldbronn, Germany) between 300 and
00 nm at 30 °C. As a blank, 3 µM P450 in 50 mM potassium phosphate buffer, pH 7.4, with
2
O) were prepared. The absorbance spectra of each sample were
5
addition of an equivalent amount of buffer instead of substrate solution was used. Each
sample was prepared and measured in duplicate. By plotting the resulting absorbance
difference (Abs386nm – Abs420nm) against the applied substrate concentration and fitting the
data with the tight binding equation using MATLAB, K values for the different steroids were
D
obtained.[31,14]
Turnover number determination. Reactions for the determination of turnover numbers were
carried out in 5 mL scale. Each reaction contained 3 µM P450, 3 µM PdR, 16 µM Pdx, 0.5 U
2
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ChemBioChem
10.1002/cbic.202000735
-1
-1
mL formate dehydrogenase from Candida boidinii, 150 mM sodium formate, 300 U mL
catalase from bovine liver, 50 µM NADH and 2 mM of the respective steroid substrate (1-6)
in 50 mM potassium phosphate buffer pH 7.4. Steroid stock solutions of 4 or 5 mM
concentration were prepared in 1.8-4.5% (w/v) hydroxypropyl-β-cyclodextrin in diH O
2
depending on the substrate. All bioconversions were carried out at 30 °C and 250 rpm for up
to 20 h. During bioconversions, samples were taken at different time points for subsequent
HPLC and GC analysis. For that, 0.25 mL of each reaction was extracted as described
elsewhere.[12] Turnover numbers were calculated based on substrate consumption and for the
period of time where the highest substrate consumption rate was observed (usually within the
first 2-3 h of reaction). Each reaction was performed in duplicate.
In contrast, in the case of steroids 10 and 11 TONs were determined based on whole-cell
conversion. When performing preparative scale reactions of 10 and 11 using frozen cells of E.
coli C43 (DE3) (pIT2cyp154C5) (pACYCcamAB), samples were taken over time and TONs
were calculated as described in the previous paragraph.
Coupling efficiency determination. NADH depletion during bioconversions of steroids by
CYP154C5 mutants, Pdx and PdR was monitored in a spectrophotometer at 340 nm (ε340
=
-1
-1
6
.22 mM cm ). Reactions of 0.7 mL total volume included 0.4 µM purified P450, 0.4 µM
-1
-1
purified PdR, 14 µM purified Pdx, 600 U mL catalase from bovine liver, 200 U mL
superoxide dismutase, 200 µM NADH and 1 mM of the respective steroid (4 or 5 mM stock
in 1.8-4.5% (w/v) of hydroxypropyl-β-cyclodextrin in diH O) in 50 mM potassium phosphate
2
buffer pH 7.4. After the NADH was completely consumed, 0.5 mL reaction mixture was
extracted as described for the whole-cell catalysis and further analyzed by HPLC and GC in
order to determine the conversion.
Analytical-scale bioconversions. In case of whole-cell bioconversions, frozen cells of E. coli
C43 (DE3) (pIT2cyp154C5) (pACYCcamAB) overexpressing Pdx, PdR and CYP154C5 were
resuspended in 50 mM potassium phosphate buffer, pH 7.4, to the desired final OD600 of 40.
All bioconversions were carried out in 1 mL scale at 30 °C and 250 rpm with addition of
-1
glucose (0.54 mg mL final conc.) for cofactor regeneration and 1 mM substrate. Substrate
stock solutions of steroids were prepared in 36% (w/v) hydroxypropyl-β-cyclodextrin in
potassium phosphate buffer, pH 7.4. In detail, stocks with a final concentration of 2.5; 3.2;
4
.2; 4.1 and 4.2 mM for substrates finasteride (7), etiadienic acid ethyl ester (8);
ethioallocholane (9); 3-deoxydehydroepiandrostendione (10) and 5α-androstan-3-one (11)
were prepared, respectively. Control reactions were carried out in parallel with E. coli C43
2
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ChemBioChem
10.1002/cbic.202000735
(DE3) (pACYCcamAB) containing only Pdx and PdR. After 20 hours of reaction, the
bioconversions were extracted for subsequent HPLC and GC analysis. For that, 500 µL of
sample was extracted twice with ethyl acetate (300 µL) and once with chloroform (250 µL).
The organic phases were combined, dried with sodium sulfate and the solvent was removed
under reduced pressure. As an exception, conversions performed with steroid 8 were acidified
with 2 M HCl previous to the extraction procedure.
Preparative-scale bioconversions. For the conversion of steroids 10 and 11, preparative-
scale bioconversions were carried out in 50 mM potassium phosphate buffer, pH 7.4, using
resting whole cells of E. coli C43 (DE3) (pIT2cyp154c5) (pACYCcamAB) at 30 °C and 250
-1
rpm with the addition of glucose (0.54 mg mL ) for cofactor regeneration. In case of substrate
1
0, resting cells were resuspended in 100 mL buffer to OD600 ~ 40, equivalent to 7.8 µM
-1
-1
CYP154C5 and an ETC activity of 8.1 U mL (1.1 U mg of total protein), as determined by
CO-difference spectra and cytochrome c assay, respectively. Similarly, conversion of
substrate 11 was performed using resting cells resuspended in 100 mL buffer to OD600 ~ 60,
-1
-1
equivalent to 4.2 µM CYP154C5 and an ETC activity of 13.8 U mL (1.4 U mg of total
protein). Initial substrate concentrations of 1 mM (10 and 11) were used; for that, stock
solutions of substrates 10 and 11 were prepared in 36% (w/v) hydroxypropyl-β-cyclodextrin
(
in diH O) and DMSO, respectively. Preparative-scale bioconversions of progesterone (3)
2
were carried out in 50 mM potassium phosphate buffer, pH 7.4, in shake flasks using resting
whole cells of E. coli C43 (DE3) (pIT2cyp154c5_F92A) (pACYCcamAB) at 30 °C and 250
rpm. In this case, cells were resuspended in 80 mL buffer to OD600 ~ 40 equivalent to 8 µM
-1
-1
CYP154C5 and an ETC activity of 4.7 U mL (0.6 U mg of total protein), as determined by
CO-difference spectra and cytochrome c assay, respectively. After 24 h of reaction, the
complete reaction volume was extracted twice with ethyl acetate (50 and 40 mL) and once
with chloroform, (30 mL), the organic phases were combined, dried with sodium sulfate and
the solvent was removed under reduced pressure. Hydroxylated steroid products were
afterwards purified by silica gel column chromatography with a mixture of ethyl acetate: n-
heptane (8:2) as mobile phase.
GC and HPLC analyses. In the case of pregnenolone (1), dehydroepiandrosterone (2),
ethioallocholane (9), 3-deoxydehydroepiandrosterone (10) and 5-androstan-3-one (11), the
solid residues after biocatalysis were redissolved in chloroform containing 30 mM cholesterol
as internal standard. In case of substrates etiadienic acid ethyl ester (8) samples were
dissolved in pure chloroform. Samples were analyzed on a GC2010 gas chromatograph
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ChemBioChem
10.1002/cbic.202000735
(Duisburg, Shimadzu) equipped with an OPTIMA 17ms column (Macherey-Nagel, Düren,
-1
Germany) with a linear gradient starting at 250 °C and heating with 10 °C min until 300 °C,
with the exception of substrate 9 were the gradient started at 230 °C. Injector and detector
temperature were set to 350 and 300 °C respectively. Substrates and products were detected
by flame ionization detector (FID). Substrates, pregnenolone (1), dehydroepiandrosterone (2),
etiadienic acid ethyl ester (8), ethioallocholane (9), 3-deoxydehydroepiandrosterone (10) and
5
3
-androstan-3-one (11) were detected with a retention time of 9.69 min, 7.94 min, 9.36 min,
.87 min, 4.97 min and 5.37 min, respectively, whereas their known products eluted at 9.27
(16-OH-1), 10.18 min (16-OH-2), 6.14 min (16-OH-10) and 8.24 min (15-OH-11),
respectively. In case of substrate finasteride (7) the solid residue was re-dissolved in
chloroform and samples were analyzed on a GC2010 equipped with a Supreme 5ms column
(CS Chromatographie Service, Langerwehe, Germany). For separation, an isothermal
temperature program at 300 °C was applied for 20 min. Injector and detector temperature
were set to 300 °C. Substrate 7 was eluted with a retention time of 5.22 min.
The dried residues of bioconversions with progesterone (3), androstendione (4), testosterone
(5) and nandrolone (6) were dissolved in acetonitrile: water (60:40) and injected on an ultra-
fast liquid chromatograph (UFLC, Prominence, Shimadzu) equipped with a Nucleosil 100-5
C18, 250 x 4,5 mm column at 50 °C. Acetonitrile: water (60:40) was used as mobile phase
-1
with a flow rate of 1.2 mL min . Detection of substrates and their respective products was
performed by UV absorbance at 243 (5 and 16-OH-5) and 242 nm (3, 4, 6, 16-OH-3, 16-
OH-4 and 16-OH-6). The substrates were eluted at 5.93 min (5), 10.45 min (3), 6.35 min (4)
and 5.39 min (6), whereas the main products were detected at 3.23 min (16-OH-5), 4.35 min
(16-OH-3), 5.60 min (21-OH-3), 3.87 min (16-OH-4A) and 3.26 min (16-OH-6A). In all
cases the conversions were calculated based on substrate consumption.
GC-MS and NMR analyses. Preliminary product identification was performed with a gas
chromatograph - mass spectrometer (GC-MS-QP2010S, Shimadzu, Germany) equipped with
an OPTIMA 17ms column (Macherey-Nagel, Germany) using a linear temperature gradient
-1
starting at 250 °C and heating with 10 °C min to 300 °C. Injector, interface and ion source
temperature were set to 300, 300 and 200 °C respectively. Structure elucidation of formed
1
13
products was performed by H-, C-, COSY, HSQC, DEPT and NOESY NMR analysis on
Bruker AV400, AV500 or AV600 instruments using deuterated chloroform or DMSO as
solvent with TMS as internal standard. Chemical shifts (δ) are given in ppm and coupling
constant (J) in Hz.
2
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ChemBioChem
10.1002/cbic.202000735
MD simulations. Molecular dynamics simulations were performed using Yasara with
algorithms that were recently described in detail elsewhere.[32] Periodic boundary conditions
were applied. Long range electrostatics, beyond 7.86 Å, were calculated through the particle
mesh Ewald method using 4th degree spline functions. The time-step was 2.0 fs, with the non-
bonded interactions updated every 2 time-steps. The force-field was Yamber3, which is an
Amber99 derivative which was specifically parameterized for structural accuracy.[33] The
compound I structure was generated and its atomic point charges assigned as described by the
Pleiss group.[34] Point charges on the steroid substrates were generated with AM1-BCC,
which gives similar accuracy as RESP at a much lower computational cost.[35]
Prior to the MD simulations, an energy minimization (described previously[36]) was used to
remove subtle steric clashes. For each of the modeled enzyme-substrate complexes, three
independent MD simulations were carried out. These simulations were started with different
initial atom velocities assigned via a random seed number.[37] The distribution of these atom
velocities always obeyed a Boltzmann distribution. In the first 30 ps of the MD simulation,
the temperature was gradually increased from 5 to 298 K. After that, the simulation was
allowed to equilibrate for 1970 ps. The subsequent production phase was 20 ns. From the
latter phase, all the reported results were collected. Snapshots were saved every 50 ps.
During the production phase of the MD simulations, geometric information was recorded to
quantify to which extent hydrogen atoms of the substrate were in a suitable orientation to be
attacked by the oxygen atom of compound I. These geometries were recorded every 1 ps, on
the fly. As suggested by the Bruice group, near attack conformations (NACs) were defined as
having interatomic distances of less than the Van der Waals contact distance and angles
between the reacting atoms within 20º of those in the quantum mechanically modeled
transition state (Figure S8).[38,39] A Yasara script that automatically performs the MD
simulations, the recording of these NACs, and analysis of the resulting data is available upon
request.
Docking. A challenge with the flexible P450 class of enzymes is that substrate binding often
requires small backbone changes. Therefore, docking was carried out essentially as previously
performed by the Reetz group for modeling steroid binding and conversion by P450 BM3
variants.[10] First the F92A mutation was introduced into the four experimentally determined
CYP154C5 structures that have a steroid substrate bound.[11] Subsequently 12 ns MD
simulations were carried out, to sample the possible backbone changes around the active site.
Progesterone was docked to snapshots of these MD simulations (1 snapshot was used per ns
of MD simulation). The docking was performed using Autodock4[40] with 4995 docking runs
2
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ChemBioChem
10.1002/cbic.202000735
for each snapshot and 25000 energy evaluations per docking run. Substrate orientations with
an unrealistic binding orientation were avoided by eliminating all poses of which the
predicted binding energy fell outside the 90% confidence interval of the substrate orientation
with the best binding energy, as described earlier.[41] The same protocol was used for docking
steroid 10 as well as 16α- or 4β-hydroxylated 10 into the active site of CYP154C5 but using
the wild-type structures as starting points.
Protein crystallography. Co-crystallization experiments for wild-type CYP154C5 with 5α-
androstan-3-one (11) were set up using a HoneyBee 961 pipetting robot (Digilab Genomic
Solutions, Hopkinton, U.S.A.) mixing 200 nL of protein solution (containing 40 mg/mL of
enzyme and 2 mM of ligand) and 200 nL of reservoir solution, and monitored with a Rock
Imager 1000 automated microscope (Formulatrix, Bedford, U.S.A.). As crystallization trails
for wild-type CYP154C5 based on literature conditions[11] did not directly yield crystals,
sparse matrix screens were applied to identify 0.2 M ammonium sulfate, 0.1 M BisTris pH 6.5
and 25% (w/v) PEG3350 as suitable precipitant mixture. Brownish, triangle-shaped crystals
were cryoprotected by soaking in reservoir solution containing 10% (v/v) 2,3-(R,R)-
butanediol prior to freezing in liquid nitrogen. 3600 images with an oscillation angle of 0.1°
were collected at beamline X06SA (PXI) at the Swiss Light Source (Paul Scherrer Institut,
Villigen, Switzerland) on an EIGER 16M X detector.
Structure determination. Data sets were processed using DIALS[42], POINTLESS[43] and
AIMLESS[44] of the CCP4 suite[45] applying a resolution cut-off of 2 Å yielding a CC1/2
higher than 0.5 in the lowest resolution shell. Initial maps were calculated by Fourier
synthesis using phenix.refine[46] and the atomic coordinates of CYP154C co-crystalized with
testosterone (PDB: 4j6d). The structure was further refined by alternating rounds of manual
adjustment in COOT[47] and computer-driven refinement with phenix.refine, including TLS
refinement. Geometry restraints for 5α-androstan-3-one were calculated using the Grade Web
Server (http://grade.globalphasing.org; Global Phasing Inc.). Data processing and refinement
statistics are listed in Supplementary Table S3. Diffraction data and coordinates were
deposited in the Protein Data Bank[48] (PDB: 6TO2).
Acknowledgements
We thank Suzanne Flaton (HIMS, University of Amsterdam) for assistance with preparative-
scale reactions. We also thank Ines Bachmann-Remy (ITMC, RWTH Aachen University) and
Jan Meine Ernsting (HIMS, University of Amsterdam) for technical assistance with NMR
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ChemBioChem
10.1002/cbic.202000735
measurements. We further acknowledge the Paul Scherrer Institute, Villigen, Switzerland for
provision of synchrotron radiation beamtime at beamline X06SA of the SLS and would like to
thank Dr. Takashi Tomizaki for assistance.
This project was financially supported by the German Research Foundation (DFG) within the
national Excellence Initiative funding scheme to promote science and research at German
universities. Additionally, financial support of JARB and TK by the German Research
Foundation via the Research Training Group PROCOMPAS [GRK 2223] is gratefully
acknowledged.
Author Contributions
AS, HJW and DBJ designed the work; PB, HJW, BN, AV, PS and JARB performed
experiments; PB, HJW, PS, TK, JARB and AS analyzed the data; PB, HJW, TK and AS
wrote the manuscript; AS, WB and DBJ revised it critically. All authors approved the final
version for publication.
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Table of contents
Changes in selectivity: Using substrate and protein engineering based on key residues and
functional groups, the regioselectivity of the bacterial cytochrome P450 monooxygenase
CYP154C5 in the hydroxylation of different steroids was altered. Apart from 16α-
hydroxylation, steroid products containing hydroxyl groups in 15α and 21 position were
formed.
Keywords: biocatalysis, steroid hydroxylation, cytochrome P450 monooxygenase, protein
engineering, regioselectivity
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