Exploring the Biocatalytic Potential of a Self-Sufficient Cytochrome P450 from Thermothelomyces thermophila

Article abstract of DOI:10.1002/adsc.201900076

Among nature's arsenal of oxidative enzymes, cytochrome P450s (CYPs) catalyze the most challenging reactions, the hydroxylations of non-activated C?H bonds. Human CYPs are studied in drug development due to their physiological role at the forefront of metabolic detoxification, but their challenging handling makes them unsuitable for application. CYPs have a great potential for biocatalysis, but often lack appropriate features such as high and soluble expression, self-sufficient internal electron transport, high stability, and an engineerable substrate scope. We have probed these characteristics for a recently described CYP that originates from the thermophilic fungus Thermothelomyces thermophila (CYP505A30), a homolog of the well-known P450-BM3 from Bacillus megaterium. CYP505A30 is a natural monooxygenase-reductase fusion, is well expressed, and moderately tolerant towards temperature and solvent exposure. Although overall comparable, we found the stability of the enzyme's domains to be inverse to P450-BM3, with a more stable reductase compared to the heme domain. After analysis of a homology model, we created mutants of the enzyme based on literature data for P450-BM3. We then probed the enzyme variants in bioconversions using a panel of active pharmaceutical ingredients, and activities were detected for a number of structurally diverse compounds. Ibuprofen was biooxidized in a preparative scale whole cell bioconversion to 1-, 2- and 3-hydroxyibuprofen. (Figure presented.).

Full text of DOI:10.1002/adsc.201900076

Title: Exploring the Biocatalytic Potential of a Self-Sufficient
Cytochrome P450 from <i>Thermothelomyces thermophila</i>
Authors: Maximilian Fürst, Bianca Kerschbaumer, Claudia Rinnofner,
Anna Migglautsch, Margit Winkler, and Marco Fraaije
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900076
Link to VoR: http://dx.doi.org/10.1002/adsc.201900076

10.1002/adsc.201900076
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Exploring the Biocatalytic Potential of a Self-Sufficient
Cytochrome P450 from Thermothelomyces thermophila
Maximilian J. L. J. Fürsta, Bianca Kerschbaumerb, Claudia Rinnofnerb,c, Anna K.
Migglautschd, Margit Winklerb, Marco W. Fraaijea*
a
Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands
Tel: (+31)-50-36-34345; e-mail: m.w.fraaije@rug.nl
Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria
Bisy e.U., Wetzawinkel 20, 8200 Hofstätten/Raab, Austria
Institute of Organic Chemistry, Graz University of Technology, NAWI Graz, 8010 Graz, Austria
b
c
d
Received: ((will be filled in by the editorial staff))
Abstract. Among nature’s arsenal of oxidative enzymes, Although overall comparable, we found the stability of the
cytochrome P450s (CYPs) catalyze the most challenging enzyme’s domains to be inverse to P450-BM3, with a more
reactions, the hydroxylations of non-activated C-H bonds. stable reductase compared to the heme domain. After
Human CYPs are studied in drug development due to their analysis of a homology model, we created mutants of the
physiological role at the forefront of metabolic enzyme based on literature data for P450-BM3. We then
detoxification, but their challenging handling makes them probed the enzyme variants in bioconversions using a panel
unsuitable for application. CYPs have a great potential for of active pharmaceutical ingredients, and activities were
biocatalysis, but often lack appropriate features such as high detected for a number of structurally diverse compounds.
and soluble expression, self-sufficient internal electron Ibuprofen was biooxidized in a preparative scale whole cell
transport, high stability, and an engineerable substrate scope. bioconversion to 1-, 2- and 3-hydroxyibuprofen.
We have probed these characteristics for a recently described
CYP that originates from thermophilic fungus
Thermothelomyces thermophila (CYP505A30), a homolog
of the well-known P450-BM3 from Bacillus megaterium.
CYP505A30 is a natural monooxygenase-reductase fusion,
is well expressed, and moderately tolerant towards
temperature and solvent exposure.
Keywords: active pharmaceutical ingredient; biocatalysis;
heme proteins; hydroxylation; ibuprofen; oxygenation; self-
sufficient cytochrome P450
Introduction
One of the most challenging reactions in organic
chemistry is the oxidation of non-activated carbons.
Overcoming the inertness of C-C and C-H bonds
requires very high reaction temperatures or highly
reactive chemical species, commonly resulting in
energy intensive and poorly controllable processes.[1]
In nature, ubiquitously found enzymes called
cytochrome P450s (CYPs or P450s) use molecular
oxygen as the oxidant to perform various oxidation
reactions (hydroxylations, dealkylations, heteroatom
oxidations and epoxidations), while forming water as
the byproduct.[2] Initially, CYPs were studied in
humans: acting as a phase I metabolic frontier, P450s
oxidize xenobiotics to increase their solubility and
activate them for further metabolism.[3] With the
majority of drugs being direct substrates, the
comprehension of CYPs is of paramount importance
in clinical and pharmaceutical science. However, the
physiologically obligatory substrate promiscuity
rendered them also as valuable biocatalysts for the
selective oxidation of non-activated carbons under
mild process conditions.[4]
The complex reaction mechanism (Scheme 1)
enabling the remarkable chemistry of P450s involves
a heme prosthetic group, whose porphyrin ring
coordinates an iron atom that is proximally ligated to
a cysteine. If a suitable substrate enters the active site,
it displaces an axial water molecule and triggers the
reductive activation of oxygen. Electrons from a
nicotinamide cofactor (NADPH or NADH) are
successively shuttled through a reductase system to
the heme. The one electron-reduced ferrous state
binds dioxygen and the second electron creates the
ferric peroxy complex. Protonation and water
elimination yields “Compound I”, which reacts with
the substrate through a radical-rebound mechanism.[2]
Unproductive pathways shortcutting this principle
cycle lead to the uncoupling of electron consumption
1
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10.1002/adsc.201900076
Advanced Synthesis & Catalysis
Scheme 1. Catalytic cycle of cytochrome P450s[5] (CYPs) and comparison of membrane-bound Class II (depicted in the
first electron transfer step) and soluble class VIII CYPs (depicted for the second electron transfer step).[6] In both classes,
the two required electrons originate from a hydride transferred from NAD(P)H. The electron acceptor is a flavin cofactor
in a cytochrome P450 reductase, which is either a separate enzyme (class II) or a fused domain (class VIII). The electrons
are successively shuttled to the heme via a second flavin, allowing first the reaction with dioxygen and then the maturation
to the catalytically active “compound I” (marked by *). Uncoupling pathways (grey dashed lines) can lead to the formation
of reactive oxygen species.
from product formation and can create hydrogen
peroxide (H2O2) or superoxide. This characteristic
can be exploited to drive the reaction reverse using
excess H2O2, leading to a cofactor-avoiding shunt
pathway.[7] In nature, most often an FAD-containing
reductase acts as the hydride acceptor, and a
ferredoxin or flavodoxin as the single electron
mediator. The individual components can exist as
separate, or as fused, multidomain proteins (“self-
sufficient”). The various possible combinations
differentiate the CYP subclasses. In bacteria, the CYP
system is typically cytosolic, while it is membrane-
bound in eukaryotes.[6] For this reason, the
physiologically relevant human CYPs, which are
membrane-bound two component systems, are
typically isolated in microsomes and their handling is
generally challenging.[8] An easier to study model
system was found in CYP102A1 (also known as
P450-BM3) from Bacillus megaterium. P450-BM3 is
soluble, all components are fused to one polypeptide,
and the single electron mediator is an FMN-
containing, flavodoxin-like domain, equivalent to the
human microsomal system.[9] Significant advances in
understanding CYP’s redox transfer mechanisms
were made with P450-BM3,[5b] but due to its
unprecedented catalytic rate, it was quickly conceived
as an outstanding biocatalyst as well.[10] After the
crystal structure was solved,[11] this notion has since
been corroborated by the accomplishments in
engineering the enzyme to work on countless
unnatural substrates. Desired activities are generally
either of interest for organic synthesis, e.g. in late
stage functionalization,[12] or mimic human drug
metabolism. The latter can substantially simplify
pharmacokinetics studies, where an access to drug
metabolites is required for adverse drug reaction
assessments or prodrug studies.[13]
Since many target compounds for P450s are
hydrophobic, organic solvent systems commonly
need to be applied. In this regard, (industrial)
application of CYPs encountered a major hurdle,
because they typically suffer from poor stability in
these conditions.[14] Besides the use of solvents,
industrial processes often run optimally at elevated
temperatures to increase reaction rates and compound
solubility. Thermo- and solvent stability of proteins
often go hand in hand and can be improved
simultaneously.[15] Although the mesophilic P450-
BM3 is already considerably more stable than e.g.
human CYPs,[16] it was engineered toward even
higher solvent- and thermostability using random
mutagenesis.[17]
However,
considerable
improvements need to be achieved for application of
a P450 biocatalyst in most industrial processes.[18]
Here we report on our investigations on a recently
characterized
CYP
from
the
fungus
2
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10.1002/adsc.201900076
Advanced Synthesis & Catalysis
Thermothelomyces thermophila
(CYP505A30).[19]
major absorption peak at 415 nm. Upon reduction and
carbon monoxide binding, the characteristic 450 nm
soret band was observed. Dodecanoic (lauric) acid
was used as a standard substrate. Fast consumption of
either NADH or NADPH was observed when
followed spectrophotometrically at 340 nm in the
presence of 1 µM of enzyme and 1 mM substrate,
confirming a catalytically competent and self-
sufficient monooxygenase with preference for
NADPH, in agreement with what was reported
recently.[19] In the absence of an organic substrate, we
measured an NADPH oxidation “leak rate” of only
0.02 s-1, significantly lower than the reported values
This enzyme is of the same class as P450-BM3 and
could emerge as an alternative biocatalyst due to its
mesophilic nature and distinct substrate scope. We
show that CYP505A30’s stability is similar to P450-
BM3’s, but with inverted stability differences
between the P450 and reductase domains. Further
characterization of the enzyme was conducted and
several substrates for the wild-type and mutated
variants were identified.
Results and Discussion
for P450-BM3, which range from 0.083 s-1 to 0.5 s-
1 [22]
.
GC-MS analysis of the ethyl acetate-extracted
Stimulated by our previous experience in identifying
biocatalytically interesting oxidoreductases in the
and TMS-derivatized reaction mix containing a
phosphite
dehydrogenase-based
NADPH
thermophilic
fungus
Thermothelomyces
regeneration system[23] confirmed a hydroxylating
activity on lauric acid. We found full conversion after
24 h and 86% ω-1 oxidation (Figure S2). A similar
result was reported by Baker et al.[19]
thermophila[20] (formerly known as Myceliophthora
thermophila[21]) we decided to focus on a gene
encoding a cytochrome P450-homolog (locus tag
MYCTH_101224). Sequence alignment revealed the
gene to consist of a single open reading frame for a
self-sufficient P450 of 1080 amino acids, identical to
CYP505A30.[19] A synthetic and codon-optimized
gene was cloned in a pET28a vector conferring an N-
terminal 6x histidine tag, and the protein was
expressed in Escherichia coli BL21 (DE3). Nickel-
Sepharose affinity chromatography yielded a protein
of approximately 125 kDa, as estimated by SDS-
PAGE (Figure S1). The red-brown protein showed a
We next assessed the thermostability of
CYP505A30 under varying conditions by
determining its apparent melting temperature (Tm).
Using a thermal-shift assay, the unfolding of the
entire protein can be monitored by supplementing a
dye that exhibits fluorescence upon binding to the
exposed hydrophobic core. The Tm is defined as the
temperature at the inflection point of the
corresponding melting curve.
Figure 1. pH profile and solvent tolerance of CYP505A30. As the enzyme exhibits two distinct unfolding events, two Tms
are plotted in (A) and (C). The higher stability curve can be assumed to correspond to the reductase domain, as it matches
the profile found when following the fluorescent flavin cofactor of that domain (Figure S3). pH profiles (A and B) were
determined in presence of 100 µM NADPH and 1 mM lauric acid, in 50 mM Na+ acetate, Na+ phosphate or Tris/HCl
buffers, indicated in shades of grey. Solvent tolerance was measured in Tris/HCl at pH 7.4 in the presence of varying
amounts of ethanol, methanol, acetonitrile and DMSO, indicated by various colors.
3
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10.1002/adsc.201900076
Advanced Synthesis & Catalysis
For CYP505A30, we observed two distinct melting
temperatures that differed by approximately 8 °C,
attributable to three-state unfolding. Using
ethanol, which decreased the Tm of both domains by
6 °C, while DMSO had the least influence and
decreased the Tm by only 1.5 °C at 10%. Moreover,
the apparent activity decreased only marginally in the
presence of cosolvents, and at comparable rates for
all solvents used.
a
principally the same assay, Baker et al. did not
observe such an apparent intermediate state, which
may be due to the different protocol in temperature
ramping. The two Tms are in good agreement,
however, for what they reported as the unfolding
temperature of the full protein (58 °C) and a truncated
protein consisting of only the heme domain (48 °C),
respectively. To establish whether the second
maximum corresponds to the reductase domain, we
performed the same assay without addition of dye,
following the fluorescence of the flavin cofactors
instead. As expected, this so-called ThermoFAD
method[24] resulted in only one peak for the Tm, which
we assigned to the flavin-containing reductase
domain (Figure S3). In contrast to P450-BM3,
CYP505A30 showed a higher stability for the
reductase domain as compared to the heme domain.
Figure 1A shows the pH-dependent melting
temperatures of the two domains of CYP505A30 in a
pH range from 3.6-9. The protein displayed a
maximal stability at slightly acidic pH, with the
maximum Tm of the reductase domain found to be
56.25 °C at pH 6, and the highest Tm of the heme
domain of 49.75 °C at pH 5.6. Although Baker et al.
reported a stability maximum at pH 7, Figure 1A
shows that CYP505A30 displays a very broad pH
stability and a barely changed Tm of the reductase
domain in the pH range 5-7.5. This stability is an
attractive feature from an application point of view
and one that also facilitates kinetic studies and the
investigation of protonation states in the electron
transfer reactions. When comparing these values to
literature data, we found them to be similar to P450-
BM3, which showed three distinct peaks of 48 °C,
58.4 °C and 63 °C in a differential scanning
calorimetry assay.[16] In the case of P450-BM3,
however, the reductase domain corresponds to the
lowest unfolding temperature. With the distinct
(potentially irreversible) unfolding event at 58.4 °C
likely being deleterious to the heme domain’s activity,
CYP505A30 and P450-BM3 basically inactivate at
the same temperatures, although with an inversion of
the unfolding order of the domains. Because stability
is not necessarily correlated to catalytic activity, we
also determined a pH profile of apparent activity by
measuring NADPH consumption in the same pH
range as before (Figure 1B). The pH range of
oxidation activity was found to be more narrow and
similar to what was described for P450-BM3.[25] In
contrast to the stability optimum, the enzyme was
found to oxidize the cofactor fastest at pH 7.4, about
1.5 pH units above the stability optimum.
In order to investigate CYP505A30’s suitability as
a biocatalyst, we next looked at the enzyme’s ability
to perform reactions of value. One reason why P450-
BM3 gained high interest in the field of biocatalysis
was the successes in engineering the enzyme to
accept pharmaceutical compounds. In the absence of
a crystal structure, we first created a homology model
of the catalytic heme domain, using YASARA
(Figure 2). P450-BM3 was automatically chosen as
the template and despite the low sequence identity of
36%, the model showed a good Z-score of -0.321.
Notably, we found a high degree of structural
conservation in the substrate access tunnel and
especially the active site: 30 of 44 residues (68%)
found in a vicinity of 10 Å around P450 BM3’s
Phe87, which is located centrally in the tunnel, are
identical. In order to investigate whether this
structural similarity would translate into selectivity
similarity, we created variants of CYP505A30 based
on P450-BM3 mutants described in literature. We
chose one of the first P450-BM3 mutants reported to
show activity on drug-like molecule – a triple mutant
targeting both the active site as well as its access
tunnel,[26] and a variant with two additional mutations
found by directed evolution, named M01 in P450-
BM3, which showed a further increased activity
towards several substrates.[27]
To further assess the stability and applicability of
CYP505A30, we also measured the Tm and NADPH
consumption in the presence of various cosolvents.
With increasing solvent levels, we found an
approximately linear decrease of stability. The effect
was relatively limited and depended on the type of
solvent. The strongest effect was measured with 10%
Figure 2. Access tunnel and active site of a homology
model of CYP505A30 (top panel), compared with P450-
BM3 crystal structure (PDB code 1FAG, bottom panel).
The protein is shown with blue (CYP505A30) or cyan
(BM3) carbons as sticks and the corresponding surfaces
are cut open at various planes (black). The heme cofactor
is pink and the central iron atom is violet.
4
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10.1002/adsc.201900076
Advanced Synthesis & Catalysis
The mutations of the two variants, their position in
comparison of the HPLC chromatograms with
authentic standards prepared and reported
recently,[28b] the major product could be identified as
8-hydroxycapsaicin, amounting to 81% of the total
products. The second most abundant product (11%)
resulted from epoxidation of the double bond.
Although producing similar relative amounts of 8-
hydroxycapsaicin, the M3x and M5x mutants
produced higher amounts of an unidentified
compound with identical mass to 8-hydroxycapsaicin,
likely the product of hydroxylation at another
position. The overall conversion yield was reduced to
73.6% and 32.6%, respectively. A reduction of the
conversion yield from 43.1% to 10.4% was also
observed for the oxidation of ibuprofen by the M3x
mutant, whereas the M5x mutant still converted
37.5%. Furthermore, in contrast to the wild type,
which produced approximately equal amounts of
three different products, M5x predominantly
catalyzed the formation of one product (77.3%).
To determine the structure of the three ibuprofen
metabolites, whole cell biooxidation was studied in a
200 ml preparative scale experiment using ibuprofen
in a concentration of 3.75 mM (~150 mg). Several
samples were analyzed by HPLC/MS over a period of
9 hours and a final total conversion level of 35% was
achieved. Notably, 32% total conversion was already
reached after 3 hours. In agreement with analytical
scale experiments, ibuprofen was oxidized to 3
metabolites, to an extent of 11% (metabolite 1), 9%
(metabolite 2) and 15% (metabolite 3), respectively
(Figure 3). According to HPLC/MS, all three
metabolites had a mass of 221 g mol-1 (ESI, neg,
SIM), indicating mono-oxidation.[31] Cell emulsions
were extracted with ethyl acetate and the metabolites
were isolated by preparative reversed phase
chromatography. Isolated products (M1-M3) were
analyzed via 1D and 2D NMR spectroscopy and were
confirmed to be 2-hydroxy-ibuprofen (M1), 3-
hydroxy-ibuprofen (M2) and 1-hydroxy-ibuprofen
(M3) (Figures 3, S4-9). These results are similar to
biooxidations of ibuprofen with CYP505X from
Aspergillus fumigatus, another homologue of P450-
BM3.[28a] In line with its activity on lauric acid,[19]
CYP505A30 preferentially oxidizes aliphatic side
chains on (sub-)terminal positions; the API screen
demonstrates, however, that oxidation is not
P450-BM3, and the homologous positions targeted in
CYP505A30 can be found in Table 1. One of the
residues is a phenylalanine located directly above the
heme (F87 in P450-BM3), which is assumed to be
critical for the acceptance of larger substrates. This
residue is conserved in both proteins, as is an active
site glutamate (BM3: E267, CYP505A3: E273)
whose exact role is still under debate.[5b] Two
residues are located at the tunnel entrance: P450-
BM3’s Leu188, whose hydrophobic nature is retained
in CYP505A3’s Ala194, and Arg47, corresponding to
the very different Gly53 in CYP505A30. The fifth
position, Gly415 (corresponding to Ala426 in
CYP505A30) has probably no influence on the
activity alterations found in P450-BM3-M01[27] and
was only added for a more realistic comparison.
Wild-type CYP505A30 and the triple and quintuple
mutants were then screened for activity on a panel of
21 active pharmaceutical ingredients (APIs) using a
previously described whole cell conversion assay.[28]
Control reactions were performed with cells
harboring the same vector lacking the P450 gene. The
compounds and the conversion results are listed in
Table 2.
Table 1. Mutations of CYP505A30 variants used in this
study.
P450-
BM3
triple
mutant
(M3x)
quintuple
mutant
(M5x)
CYP505A30 mutated
residue
to
residue
Arg47
Phe87
Leu188
Glu267
Gly415
Gly53
Phe93
Ala194
Glu273
Ala426
Leu
Val
Asp
Val
Ser
x
x
x
X
x
x
x
x
Interestingly, we found that the mutations only
slightly affected the substrate scope of CYP505A30,
while they had a distinct effect on the product
distribution. All three variants oxidized eight
compounds, of which the two best substrates were
capsaicin and ibuprofen. Capsaicin is the pungency-
causing compound in chili peppers, and is undergoing
clinical trials as a therapeutic agent for various
diseases.[29] Ibuprofen is a synthetic, nonsteroidal
anti-inflammatory drug that finds widespread
application as an over-the-counter analgesic.[30]
Structurally, the two molecules show some similarity,
as they are both composed of an aromatic ring and an
aliphatic chain with a terminal isobutyl moiety (Table
2). Other compounds that were converted by all
variants were the structurally diverse phenacetin, 2-
naphthol, and tolbutamide. Phenacetine as well as
chlorzoxazone was only converted in considerable
amounts by the wild type, while diclofenac was only
accepted by the mutants. For those cases, the reaction
occurred selectively and yielded only one product.
Lidocaine and piperine were converted only in trace
amounts by all three variants. Capsaicin was most
efficiently converted by the wild-type enzyme, with
94.7% of the starting material being turned over. By
restricted to such a structural element.
Figure 3. Preparative scale whole cell biooxidation of
ibuprofen by CYP505A30. Reaction volume: 200 ml;
substrate concentration: 3.75 mM. Conversion of ibuprofen
gave three products (M1-M3) according to HPLC/MS. The
sum of M1 to M3 represents the overall total conversion
5
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10.1002/adsc.201900076
Advanced Synthesis & Catalysis
yield. The three monooxygenation products (221 g mol-1;
ESI, neg, SIM) are 2-hydroxy-ibuprofen (M1), 3-hydroxy-
ibuprofen (M2) and 1-hydroxy-ibuprofen (M3) according
to NMR (Figures S4-9).
Table 2. Conversion of APIs by CYP505A30.
a) MW = Molecular weight
b) Products are depicted as percentages in various colors, unconverted substrate is depicted white. T = trace amounts
converted.
c) Progesterone: R1,R3 = H, R2 = CH3; Hydrocortisone: R1 = OH, R2 = CH2OH, R3 = OH
Conclusion
heterologously expressed in soluble form. The
enzyme shows a certain similarity to the well-
established P450-BM3, although the stability of the
two enzyme subdomains is inverted. CYP505A30’s
more stable reductase domain could therefore be used
to generate a P450-BM3 chimera with improved
CYP505A30 could serve as a useful biocatalyst as
it combines attractive features for application: it is a
self-sufficient
enzyme that is moderately thermostable, shows low
susceptibility to solvent exposure, and it can easily be
monooxygenase-reductase
fusion
thermostability, as
has
been demonstrated
6
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10.1002/adsc.201900076
Advanced Synthesis & Catalysis
previously.[32] It’s broad pH-tolerance could overnight culture was used to inoculate a main culture of
furthermore prove useful in mechanistic or terrific broth (TB) medium which was shaken in an
crystallographic studies. Its apparent high tolerance to Erlenmayer flask without baffles at 37 °C until an OD600 of
co-solvents is a particularly useful feature for approximately 1.8 was reached. Protein expression was
biooxidation of drugs, which are often large, then induced by addition of 1 mM IPTG, upon which the
hydrophobic molecules with poor water solubility. culture was shifted to 24 °C, where it was allowed to grow
The basal activity on a surprisingly diverse range of for another 18 h. Cells were harvested by centrifugation
compounds already found for the wild-type advocates and either frozen at -20 °C or processed directly.
CYP505A30’s use as a starting point for directed After resuspension in 50 mM Tris/HCl pH 7.4, cells were
evolution. Alternative enzymes are useful for early sonicated for 15 minutes and cell debris was removed by
screens, as enzyme engineering is much more centrifugation. The cell free extract was loaded on a pre-
successful in expanding an initial (small) activity than equilibrated gravity flow column containing appropriate
introducing one from scratch.[33] (Semi)random amounts of Ni2+ Sepharose HP (GEHealthcare). The
mutagenesis and high-throughput activity screens to column was incubated at 4 °C rotating for 1 h and the flow
boost activity proved highly successful for P450- through discarded. The column was washed with 50 mM
BM3,[4, 5b] but our results suggest that a direct transfer Tris/HCl pH 7.4 and 50 mM Tris/HCl pH 7.4 containing 5
of activities may not be achieved by simply copying mM imidazole. The protein was then eluted by washing the
mutations. As wild-type CYP505A30 already column with 50 mM Tris/HCl pH 7.4 containing 500 mM
accepted several large drug-like molecules, it is imidazole. Salt was removed by applying the solution to a
maybe less surprising that our mutations had a pre-equilibrated Econo-Pac 10DG desalting column (Bio-
smaller effect as anticipated. The results nevertheless Rad). For storage, 10 % glycerol was added, the protein
demonstrate that besides substrate scope, active site shock-frozen in liquid nitrogen and stored at -80 °C.
mutagenesis can especially steer the product
Spectra
specificity.
CO-difference spectra were obtained by following the
[36]
protocol of Guengerich et al.
Jasco spectrophotometer.
and recorded on a V-660
Experimental Section
Materials
NADPH consumption assay
All standard reagents were purchased from Sigma Aldrich
unless otherwise stated.
The reaction mix of 200 µL was buffered in 50 mM
Tris/HCl pH 7.4 and contained 1 µM purified P450, 1 mM
of lauric acid (except for the uncoupling measurement) and
100 µM NADPH (or NADH in an initial experiment). The
assays were performed at room temperature. After addition
of the cofactor, the mix was transferred to a cuvette and the
reaction followed at 340 nm on a spectrophotometer (V-
660 Jasco).
Plasmid generation
A synthetic and codon optimized gene of a NCBI genbank
entry XM_003663599 was ordered from GenScript (New
Jersey, USA). The gene was ordered to be cloned via
HindIII and XhoI in a pET28a vector with kanamycin
resistance such that an N-terminal 6x-Histidine tag was
translationally fused to CYP505A3. Plasmid integrity was
confirmed by sequencing (GATC-Biotech, Germany).
Bioconversion with purified enzyme and GC-MS
analysis.
The bioconversion mix of 1 mL with purified enzyme was
prepared in 50 mM Tris/HCl pH 7.4 and contained 1 µM
purified P450, 1 mM of lauric acid and 100 µM NADPH.
For cofactor regeneration, 1 µM purified thermostable
phosphite dehydrogenase[23] and 10 mM phosphite were
added. The mix was incubated at 30 °C in a closed 20 mL
glass vial and mildly agitated for 24 h. The reaction was
stopped by adding NaCl saturated 1 M HCl and
subsequently equal amounts of ethyl acetate were added.
After thorough mixing and centrifugation, the organic layer
was removed and dried over anhydrous magnesium sulfate.
1% TMS in BSTFA was added and the sample incubated at
75 °C for 30 min. 1 µl was then injected in a GC-MS
QP2010 ultra instrument (Shimadzu) with electron
ionization and quadrupole separation, using an HP-1
column (Agilent, 30 m x 0.32 mm x 0.25 μm). The settings
of the GC oven were 30 °C, wait 5 min, 5 °C/min ramp to
70, wait 5 min, 5 °C/min ramp to 130 °C, wait 5 min,
15 °C/min ramp to 325 °C, wait 7 min.
Mutagenesis
The triple and quintuple mutants were generated using the
multichange isothermal mutagenesis method.[34] Briefly,
fully complementary forward and reverse primers were
designed at the mutated position that contained the
mutation. PCR fragments from one position to the next
were then created by combining forward and reverse
primers of adjacent positions. The fragments were then
assembled by Gibson cloning,[35] using an in-house
prepared reaction mix. The assembled plasmid was
transformed, and a re-isolated plasmid from a single colony
was sent for sequencing to confirm the presence of the
mutations.
Enzyme expression and purification
E. coli BL21 (DE3) competent cells were transformed with
the pET28a-6xHis-CYP505A30 vector (or the mutated
version) and the cells were plated on a Luria-Bertani (LB)
agar plate containing 50 µg/mL kanamycin. A single
colony was picked and grown over night in LBKan. The
Tm determination using a fluorescent shift assay
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900076
Advanced Synthesis & Catalysis
To determine the apparent melting temperature (Tm) in combined organic layers was removed under reduced
different conditions, duplicate samples of 25 µl were pressure. Ibuprofen was separated from the metabolites by
prepared in a 96-well thin-walled PCR plate. The samples silica gel chromatography. Metabolite compounds were
contained 1 mg/ml purified enzyme. The plate was heated separated via reverse phase HPLC on a Thermo Scientific
from 20 °C to 90 °C, increasing temperature by 0.5 °C Dionex Ulti Mate 3000 Instrument using a Macherey-
every 10 seconds, using an RT-PCR machine (CFX96- Nagel VP 125/21 Nucleodur 100-5 C18 EC column.
Touch, Bio-Rad Laboratories) that measured fluorescence A linear gradient of 2% to 65 % MeCN in 24 min was used.
using a 450–490 excitation filter and a 515–530 nm 1D and 2D NMR spectroscopy (1H, 13C, APT, COSY,
emission filter. The melting point was defined as the HSQC, HMBC) were recorded on a Bruker AVANCE III
inflection point of the resulting melting curve, equivalent to 300 spectrometer with autosampler (1H: 300.36 MHz; 13C:
the maximum of the first derivative of the same curve.
75.53 MHz) and chemical shifts are referenced to residual
Three different buffers at 50 mM concentration were used protonated solvent signals as internal standard. To facilitate
to cover a wide pH range: sodium acetate for pH 3.6-5.6, the interpretation, the C-spectra were proton decoupled to
sodium phosphate for pH 5.8.-8.0, and Tris/HCl for 7.4-9.0. gain better identification of the peaks.
Samples containing co-solvents were in 50 mM Tris/HCl
Homology model
pH 7.4.
The homology model of CYP505A30 was created using
YASARA version 15.11.18. The amino acid sequence in a
Activity assay in varying conditions
The same plate as for the Tm determination was prepared FASTA format was used as an input, and the program’s
and diluted in a 96-well microtiter plate with the same hm_build.mcr macro was used with standard settings.[37]
buffer layout to achieve an enzyme concentration of 1 µM The model was mainly built on the P450-BM3 structure
in each well. Activity was measured in a plate reader with pdb code 1ZO9[38], while some parts were the result of
(Synergy MX microtiter plate reader, BioTek Instruments) hybridization with models built based on pdb codes 2IJ3,
with automatic dispensing of NAPDH (final concentration 3KX3, and 1ZO4. The overall quality Z-score of the model
100 µM) and lauric acid as a substrate (final concentration was -0.321, which is judged “good” by YASARA.
1 mM) before measuring absorbance at 340 nm for 1 min.
Whole cell conversions of APIs
Acknowledgements
Frozen cells were thawed and resuspended in potassium
phosphate buffer (100 mM pH 7.4, 8.5% w/v sucrose) in
Tanja Kainz is acknowledged for technical support. The research
for this work has received funding from the European Union (EU)
24-well plates. Trisodium citrate (50 µL, 1 M in ddH2O),
NADP+ (50 µL, 1 mM in ddH2O), MgCl2 (10 µL, 1 M in
ddH2O), glucose (10 µL, 1 M in ddH2O), and GDH (5 µL,
10 mg mL-1 in ddH2O) were added to 825 µL of cell
suspension. The reaction was started by the addition of
substrate (50 µL, 100 mM in DMSO). The final cell
density at 600 nm (OD600) was 100 in a total volume of 1
mL. The plate was sealed with gas permeable adhesive seal
and incubated at 30 °C and 120 rpm in an orbital shaker for
16 h. The reactions were terminated by the addition of
MeOH/ACN (1 mL 1:1 v/v). After mixing, the reaction
mix was centrifugation in 1.5 mL Eppendorf. (5 min, 4 °C,
13,400 rpm). 200 µL of the supernatant were analyzed in
polypropylene microtiter plates by HPLC-MS on a 1200
HPLC Series equipped with G1379B degasser, G1312B
binary pump, SL G1367C HiP-ALS SL autosampler,
G1314C VWD SL UV detector, G1316B TCC SL column
oven and G1956B mass selective detector (MSD) with a
Kinetex 50x4.6 mm; 2.6u; C18; 100 An HPLC column
(Phenomenex) equipped with a UHPLC C18 Security
Guard ULTRA cartridge (Phenomenex).
project ROBOX (grant agreement n° 635734) under EU’s
Horizon 2020 Programme Research and Innovation actions
H2020-LEIT BIO-2014-1.
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9
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Advanced Synthesis & Catalysis
FULL PAPER
Exploring the Biocatalytic Potential of a Self-
Sufficient Cytochrome P450 from
Thermothelomyces thermophila
Adv. Synth. Catal. Year, Volume, Page – Page
Maximilian J. L. J. Fürsta, Bianca Kerschbaumerb,
Claudia Rinnofnerbc, Anna K. Migglautschd, Margit
Winklerb, Marco W. Fraaijea*
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