VpStyA1/VpStyA2B of variovorax paradoxus EPS: An aryl alkyl sulfoxidase rather than a styrene epoxidizing monooxygenase

Article abstract of DOI:10.3390/molecules23040809

Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) doma

Full text of DOI:10.3390/molecules23040809

molecules
Article
VpStyA1/VpStyA2B of Variovorax paradoxus EPS:
An Aryl Alkyl Sulfoxidase Rather than a Styrene
Epoxidizing Monooxygenase
Dirk Tischler 1,2,* , Ringo Schwabe , Lucas Siegel , Kristin Joffroy , Stefan R. Kaschabek ,
ID
1
1
1
1
Anika Scholtissek and Thomas Heine 1
1
ID
1
Institute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29,
9599 Freiberg, Germany; ringoschwabe007@gmail.com (R.S.); lucasbenedikt@aol.com (L.S.);
0
kristin.friebel@student.tu-freiberg.de (K.J.); stefan.kaschabek@ioez.tu-freiberg.de (S.R.K.);
anika.scholtissek@gmail.com (A.S.); heinet@tu-freiberg.de (T.H.)
Microbial Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44780 Bochum, Germany
Correspondence: dirk.tischler@rub.de; Tel.: +49-234-32-22656
2
*
Academic Editor: Willem van Berkel
Received: 16 March 2018; Accepted: 1 April 2018; Published: 2 April 2018
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Herein we describe the first representative of an E2-type two-component styrene
monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a
two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain.
It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as
−
1
NADH:FAD-oxidoreductase. A K
determined and resulted in a catalytic efficiency (kcat
NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated.
m
of 33.6
±
4.0
µ
M for FAD and a kcat of 22.3
±
1.1 s were
−1
M . To investigate its
−
1
−1
K
m
) of 0.64 s
µ
−
1
−1
−1
) while
One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcat
K
m
of 5.21 s
µM
keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1,
−
1
−1
showed monooxygenase activity on styrene of 0.14 U mg and 0.46 U mg , as well as on benzyl
−
1
−1
methyl sulfide of 1.62 U mg and 3.11 U mg , respectively. The high sulfoxidase activity was
the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high
substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of
all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity.
In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase
activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as
well as on the monooxygenase performance. Overall, this monooxygenase represents a promising
candidate for biocatalyst development and studying natural fusion proteins.
Keywords: sulfoxidation; epoxidation; two-component monooxygenase; flavoprotein; enantioselective
biotransformation; fusion protein; protein linker; soil microorganism
1
. Introduction
Flavin-dependent monooxygenases are able to catalyze a number of biotechnologically important
reactions which are often regio- and enantioselective [
eight groups according to their structure and function [
and sulfoxidation reactions are attractive for many purposes [
monooxygenases (SMOs) represent a group performing both reactions with a certain selectivity [
This is group E among the flavin-dependent monooxygenases which can be described as follows
EC 1.14.14.11) [ ]. These are two-component systems. A strictly NADH-dependent oxidoreductase
1
1
–
3
]. These enzymes can be divided into
]. Regio- and enantioselective epoxidation
11]. Flavin-dependent styrene
10].
4
–
2,5–
(
1
Molecules 2018, 23, 809; doi:10.3390/molecules23040809
www.mdpi.com/journal/molecules

Molecules 2018, 23, 809
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(StyB; EC 1.5.1.36) produces reduced FAD for the monooxygenase component (StyA, StyA1). Some
of these reductases occur as self-sufficient natural fusion proteins (or two domain proteins; StyA2B)
composed of an oxygenase (A2) and reductase (B) domain. And those represent the prototype of
E2-type SMOs which were first described from a Rhodococcus [
5
]. Most of the monooxygenases
(StyA, StyA1) described utilize styrene as major substrate and even belong to natural styrene
degradation pathways [11,12]. Recently, some E2-type SMOs have been discussed as initial
step of indole detoxification or degradation [13
monooxygenases (IMOs).
,14]. Thus they can also be described as indole
E-type monooxygenases rely on the flavin cofactor flavin adenine dinucleotide (FAD).
The reduced flavin is transferred by diffusion or by direct transfer between both components [15 17].
The monooxygenase binds tightly the reduced cofactor and the oxygen driven catalysis gets
initiated [15 19]. Here oxygen is activated by the reduced FAD to a (hydro)peroxy-FAD which
can attack the actual substrate, e.g., styrene. Upon substrate oxygenation a hydroxyl-FAD intermediate
–
–
is formed and decomposes to oxidized FAD and water [18
and another catalytic cycle can start.
–20]. The product is subsequently released
E2-type monooxygenases have been shown to be excellent in sulfoxidation with respect to
substrate conversion and enantioselectivity whereas the E1-type only shows a high activity at low
enantioselectivity [
and it has a rather low activity [
monooxygenases which can be applied in biocatalysis.
Based on former studies of E2-type SMOs [4–6,9,11,21,22] it was reasonable to investigate the
1–
10]. However, so far there is only a single E2-type SMO in detail described
5,6,9]. Therefore, it is reasonable to screen for additional E2-type
phylogenetic more different system VpStyA1/VpStyA2B of Variovorax paradoxus EPS (accession
numbers: ADU39063 and ADU39062). The general activity and capability to convert styrene but
also sulfides in comparison to other monooxygenases is presented (Scheme 1).
Scheme 1. A view on styrene monooxygenase activity. The reductase domain of StyA2B reduces FAD
(displayed in oxidized form between protein monomers) upon NADH-consumption. Reduced FAD
can be used by both monooxygenase units, StyA1 (major; right) and StyA2 (minor; left), to activate
molecular oxygen and to subsequently monooxygenate the substrates (here for example styrene and
benzyl methyl sulfide) [5–10]. Upon substrate oxygenation hydroxyl-FAD is formed and thereof water
is eliminated to recycle the FAD in its oxidized form for the next catalytic cycle.
2
. Results and Discussion
2
.1. Evolution of VpStyA1/VpStyA2B from Strain EPS
During an earlier study the putative genes encoding for the monooxygenase VpStyA1/VpStyA2B
and the respective gene cluster of strain EPS were identified [21]. According to a phylogenetic analysis
and to the surrounding genomic region the proteins were assigned as a two-component SMO related to

Molecules 2018, 23, 809
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the E2-prototype of Rhodococcus opacus 1CP [
5,6
,11]. Interestingly, the sequence similarity of VpStyA1
and VpStyA2B (74% identity over 404 amino acids) between both monooxygenase domains (A1 and A2)
was much higher as among other StyA1/StyA2B systems. Furthermore, they form together a separate
branch in a phylogenetic distance tree of monooxygenase components [21,23]. This phylogenetic
study was now refined due to the release of more putative StyA1/StyA2B-sequences, especially from
Variovorax species. This can help to identify the nature and position of the linker within the two domain
proteins (StyA2B-like). The linker connects the monooxygenase (A2) and the FAD-reductase (B)
domain (Figure 1). Respectively, the fusion event was discussed as functionally convergent event [21].
However, no activities of these putative E2-type SMOs originating of Variovorax species were reported
until now.
Figure 1. (Top) Sequence alignment of StyA2B-like proteins in comparison to StyA1 as well as functional
StyA (left) and StyB (right) components. (Bottom) homology model of VpStyA1 (darkgreen) VpStyA2B
(monooxygenase—lightgreen, reductase—blue). The proposed linker region is illustrated in red.
Numbering on top is according to VpStyA2B and the terminal amino acids are given at the end of
lines. (ADU_V. paradoxus EPS; KIQ_V. paradoxus MEDvA23; SDZ_Variovorax sp. YR266; SFQ_Variovorax
sp. OK605; ACR_R. opacus 1CP; ABM_Paenarthrobacter aurescens TC1; BAD_Nocardia farcinica
IFM 10152; ABB_Pseudomonas putida SN1 (the A-component comprises the sequence GQWCSQY);
CAB_P. fluorescens ST; AAC_P. taiwanensis VLB120; ABV_uncultured bacterium; ABQ_uncultured
bacterium/MoxY; ADE_Pseudomonas sp. LQ26).
Mining the databases for styA2B-like genes/proteins revealed that they occur mainly among
Actinobacteria (e.g., Amycolatopsis, Arthrobacter, Gordonia, Mycobacterium, Nocardia, Paenarthrobacter,
Paeniglutamicibacter, Pseudarthrobacter, Sciscionella, Sinomonas, Streptomyces, among others), but also
among Variovorax (date of BLAST search: 19 September 2017; GenBank release 222). Variovorax belongs
to the class of β-proteobacteria and not to Actinobacteria. There are no styA2B-like genes found in other
none-Actinobacteria. However, there exist other styA/styB-genes encoding for protein components
with a high sequence similarity to this E2-type SMOs among various genera. The closest homologues
to Variovorax two domain proteins are found in Delftia although no natural fusion variant is present.
Already with the first report of RoStyA2B the linker region was discussed [
5]. This can now be
done with more emphasis on various sequences by means of sequence alignments as well as molecular

Molecules 2018, 23, 809
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modelling (Figure 1). A less conserved region/loop was identified as potential linker region. It is
localized at the C-terminal site of the monooxygenase domain (A2) of StyA2B-like proteins. In case
of VpStyA2B it is located around region 408-AREAV-412. The alignment of StyA2B-, StyA1- and
StyA-like amino acid sequences indicates that the last conserved amino acid among those proteins is
at position 404 (VpStyA2B) and in case of the none-fused proteins the following C-terminal sequences
are variable. In case of the two domain proteins next to this region the domain of the NADH:FAD
oxidoreductase (B-part) is localized. There the first conserved amino acid is present at position 417
(VpStyA2B) with respect to related StyA2B- and StyB-like proteins. This region from 404 to 417
represents a flexible loop between two helices according to homology modelling. This is not surprising
as there is no sequence-structure relation available for this part. However, this is in congruence
to earlier made observations in which the N-terminal sequence of the FAD-reductase domain was
investigated [9,20,21,24].
Adjacent to the monooxygenase domain (A2) and the proposed linker region the reductase
domain (B) of the natural fusion proteins follows. Interestingly, the reductase sequence misses in all
cases a few amino acids (range: 3 to 14) when compared to the StyB-like reductases of E1-type SMOs
(
Figure 1) [5,7]. The mentioned linker region was now chosen as a target for site-directed mutagenesis.
In all cases the original sequence (AREAV) was replaced or even extended. The following mutants were
successfully prepared and verified by sequencing: TIVVV, AAAAA, HHHHH, WYHHH, WYHHHHH,
and GQWCSQY. In order to validate the made assumptions, the wildtype protein VpStyA2B and
the mutant proteins were produced and assayed. The chosen linker sequences of mutants base on
the following rationale. Linker with A/V-rich sequences or H-rich were chosen to allow production
of either more flexible or more rigid linker sequences, respectively [25
,26]. Short H-rich sequences
tend to form –helical non-flexible structures [25 27]. This is well established for histidine-tagged
α
,
proteins to allow a subsequent Ni-affinity purification. Often G/S-rich linker sequences are introduced
between the H-tag and the target protein sequence to have more flexibility. The 408-GQWCSQY motif
represents the C-terminal sequence of a StyA-protein originating from Pseudomonas (Figure 1; accession
number: ABB03727) [28]. It was chosen to compare it to earlier made fusion proteins [19]. A more
recent study reports on the generation and catalytic properties of an artificial E1-type SMO fusion
protein comprising a long flexible linker [29]. This seemed to be promising for catalysis, but, does not
resemble the naturally occurring fusion proteins.
2
.2. Molecular Genetic Work and Enzyme Production
The cloning of both genes, VpstyA1 and VpstyA2B, as well as the generation of mutants was
successfully accomplished which was verified by sequencing the inserts of gene expression plasmids
(See supporting information, Table S1) and by a simple indole based activity assay.
E. coli BL21 allows the formation of indole from tryptophan during growth on complex
medium. Thus, the activity of styrene monooxygenases and related enzymes can be verified by
indole transformation to yield indigo [ 10 11 20]. Indeed, all clones obtained [E. coli BL21 (DE3)
4–6, , ,
pLysS derivatives harboring the wildtype or mutant genes in a pET-vector] produced indigo during
cultivation even without being induced for overexpression of target proteins. Clones with highest
indigo formation rate were selected, propagated and stored as glycerol stocks for later protein
production and characterization.
In both cases (VpStyA1 and VpStyA2B), enzyme production was successfully achieved with
a yield of 2 to 4 mgVpStyA2B and up to 9 mgVpStyA1 protein per liter broth, respectively. This is in
congruence to other studies [5,9,18]. In case of the mutants protein yields were significant lower.
2
.3. Reductase Activity of VpStyA2B and VpStyA2B-Mutants
The fusion protein VpStyA2B of strain EPS was supposed to be mainly a reductase of the complete
monooxygenase system [6,7], and was for those reasons characterized in analogy to the enzyme
RoStyA2B of strain 1CP [5].

Molecules 2018, 23, 809
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The protein VpStyA2B was successfully produced (verified by SDS-PAGE, see supplemental
material). The fraction obtained from Ni-chelate chromatography was slightly yellow, which is an
indicator of a bound flavin. This was analyzed as described for other SMOs. FAD was determined
by means of RP-HPLC as well as by spectroscopic methods and the use of authentic standards (see
Supplemental Material). A FAD saturation of 4.9 to 20.8 mol% was calculated for the protein applied.
This is in congruence to results obtained earlier for RoStyA2B [6,18]. This fraction was immediately
◦
assayed for activity and then concentrated and stored at
−
20 C in a suitable storage buffer until
characterization. It used NADH as source of reducing equivalents in order to reduce flavins. NADH
could not be replaced by NADPH. In case of the flavins FAD, FMN and riboflavin can be acceptors
of reducing equivalents (Table 1). However, a clear preference was not determined with respect to
−
1
−1
catalytic efficiency which was for all employed flavins between 0.57 and 0.88 s µM .
In contrast to the monooxygenase part (StyA2), promiscuity towards the flavin cosubstrate is in
accordance with most characterized SMO reductases. So far, only one representative from strain 1CP
(RoStyB) is reported to be specific for FAD [9,23,24,30].
Table 1. NADH:flavin oxidoreductase activity of VpStyA2B (MW = 66.32 kDa which was calculated
from the amino acid sequence including the N-terminal tag).
1
2
−1
−1
−1 −1
−1
Donor /Acceptor (µM)
Km (µM)
Vmax (U mg
)
kcat (s
)
kcat Km
(s µM )
NADH (7.9–164)/FAD (70)
NADH (164)/FAD (6.3–78.8)
NADH (164)/FMN (4.1–90.2)
24.0 ± 4.0
33.6 ± 4.0
45.9 ± 6.8
37.7 ± 7.2
16.2 ± 0.8
20.2 ± 1.0
26.0 ± 1.8
31.3 ± 2.7
17.9 ± 0.9
22.3 ± 1.1
28.7 ± 1.9
34.6 ± 2.9
2
0.72
0.64
0.57
0.88
NADH (164
µM)/Riboflavin (6.3–88.2)
1
NADPH (230
µM in presence of 70
µ
M FAD) did not serve as an electron donor. Either electron donor or acceptor
was present in excess and the data obtained of triplicates were analyzed assuming Michaelis-Menten kinetics.
The reductase activity and catalytic efficiency of VpStyA2B is up to 10-times higher than reported
for the Rhodococcus enzyme RoStyA2B [ ]. Thus, differences at the amino acid level (57% identity)
5
are reflected within the biochemical properties of StyA2B-like proteins. Still, the activity of the two
domain protein is by an order of magnitude lower compared to most StyB-like reductases of other
two-component systems [23,24,30]. Recently, it was reported that an N-terminal fusion for the StyB-like
proteins drastically decreases the oxidoreductase activity [24]. In addition, an artificial fusion protein
was constructed from two-component E1-type monooxygenase components of Pseudomonas [20].
Herein, the coupling (catalytic efficiency) was improved and the catalytic mechanism changed. It was
also shown that the N-terminal region of the reductase influences the binding and affinity for the
substrate [20,23]. Therefore, it is likely that the same is true for VpStyA2B. However, it is likely that
this effect of the fusion to StyA2 is different in dependence of the linker region and sequence in the
Variovorax two domain enzyme.
VpStyA2B was tested for inhibition or activation by a number of compounds used in other SMO
studies (see supplementary material). The effects of the herein tested compounds are similar as
observed for RoStyA2B [5]. It can be concluded, that there is no metal dependency present. Moreover,
3+
in contrast to RoStyA2B, the activity is lowered by addition of Fe-ions, while Fe has a stronger effect.
This is in accordance with inhibition studies on the reductase StyB from the two-component systems
of Pseudomonas taiwanensis VLB120 and Acinetobacter baylyi ADP1 [23
,
30]. However, reducing agents
as DTT and divalent ions as Ca2 support the performance of VpStyA2B. This was also observed for
RoStyA2B as well as AbStyB. Interestingly, thioanisole decreases the reductase activity, which was not
observed for SMO-reductases before.
+
In order to investigate the linker region of this two domain protein efforts to determine the
respective region were accomplished on sequence level (see above) and served for the direction in a
mutagenesis study. The six mutants of VpStyA2B obtained were separately produced and purified as
described above and characterized in analogy to the wildtype for their NADH:FAD oxidoreductase

Molecules 2018, 23, 809
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activities (Table 2). During expression also these clones produced significant amounts of indigo which
indicated a functional expression of respective mutants.
Table 2. NADH:flavin oxidoreductase activity of VpStyA2B-mutants and -wildtype.
VpStyA2B-Variant
Donor NADH 1
Acceptor FAD 1
Vmax (U mg 1)
−
kcat Km
−1 −1
(s µM
−1
)
Km (µM) Vmax (U mg
−1
)
kcat Km
−1 −1
(s µM )
−1
Km (µM)
wildtype VpStyA2B
24.0
37.7
28.1
44.2
20.1
40.6
47.6
16.2
3.3
8.9
2.5
7
0.72
0.09
0.34
0.06
0.37
0.36
0.09
33.6
5.1
1.8
14.5
3.2
7.4
20.2
3.1
8.8
3.1
6.9
0.64
0.65
5.21
0.23
2.30
1.90
0.27
408-TIVVV
408-AAAAA
4
4
08-HHHHH
08-WYHHH
408-WYHHHHH
13.8
3.8
13.2
3.7
408-GQWCSQY
14.5
1
In order to determine kinetic properties concentrations of NADH and FAD were chosen as for the wildtype
VpStyA2B given in Table 1. Triplicates were used to get the values and standard deviations were in all cases less
than 15% and comparable to Table 1.
All mutants were active and it was possible to determine specific activities (Table 2).
Maximum specific activities were determined as described in materials and methods according to
Michaelis-Menten. Excess of FAD did not change these values, but had to be assayed as the protein
preparations might have different FAD-saturations as it was found for the wildtype two domain
proteins [
6
,18]. The wildtype was most active with NADH and all the mutants showed a lower activity.
However, the affinity for NADH seemed not to be altered as the K
m
values were all in the same high
range. That changed for FAD. The general activities were similar to those for NADH-variation and
also the order from the most active to the worst representative was the same. But, the affinity for FAD
was drastically different among the variants which was obvious from the K values determined and
m
also reflected by the catalytic efficiencies (Table 2). The most efficient variant was AAAAA-mutant
with respect to its oxidoreductase activity. Respectively, the catalytic efficiency of FAD-reduction was
−
1
−1
determined to 5.21 s
µM
which is about 8.1-times more efficient than the wildtype. This is due to
value for FAD. In addition, the mutants
the tighter FAD binding expressed by the 18.7-times lower K
m
WYHHH and WYHHHHH showed an increased catalytic efficiency while the mutants HHHHH and
GQWCSQY showed a decreased catalytic efficiency. Interestingly, mutant TIVVV was as efficient as
the wildtype two domain protein but had a lowered specific activity.
Hence, the proposed and mutated linker region has a strong effect on the oxidoreductase activity.
This might be due to a changed communication of domains (A2 and B) or due to altered FAD binding
and turnover. This is in congruence to earlier studies which demonstrated that the N-terminal part
of NAD(P)H:flavin oxidoreductases is important for the flavin binding and thus for catalysis [20,24].
Further, it indicates that the selected sequence region indeed can be the linker as it is functional relevant
for the reductase domain.
2
.4. Monooxygenase Activity of VpStyA1, VpStyA2B and VpStyA2B-Mutants
During the gene cloning and expression experiments the formation of indigo was observed and
used to identify best protein producers. This was verified by a plate assay in order to get a view on
the strains producing either a highly active SMO or a lot of protein (Table 3). The wildtype proteins
VpStyA1 and VpStyA2B and especially the AAAAA-mutant produced most indigo. The acceptance
of indole as a substrate and the formation of indigo fits to very recent findings that related E2-type
two-component monooxygenases were assigned to indole degradation and act basically as indole
monooxygenases [13,14].
The wildtype monooxygenase components VpStyA1 and VpStyA2B show a comparable epoxidase
−
1
activity as other SMOs (range 0.02 to 2.1 U mg ) (Table 3) [5–10,30]. VpStyA1 is about 2.9-times and
.1-times more active than VpStyA2B and the Rhodococcus counterpart RoStyA1, respectively. And it
was confirmed that VpStyA1 represents the major monooxygenase of the system VpStyA1/VpStyA2B
as it was found for the prototype RoStyA1/RoStyA2B [ ]. This was expected but also indicates that the
2
6

Molecules 2018, 23, 809
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high sequence similarity of the Variovorax monooxygenases does not change the catalytic properties to
a large extent. However, VpStyA1/VpStyA2B does not reach the high epoxidase activity of StyA of
−
1
strain VLB120 (2.1 U mg ) [30].
Table 3. Epoxidase activity of VpStyA2B-mutants and -wildtype enzymes.
Styrene
Substrate
SMO 1
Indole
Plate Screening 4
−1
ETY 2 (%)
Vmax (mU mg ) + Extra RoStyBart 3
−1
Vmax (mU mg
)
wildtype VpStyA1
wildtype VpStyA2B
n.a.
159
135
260
<1
1.7
<1
<1
n.a.
1
4.4
3
<1
<1
<1
<1
460
140
327
260
4
7.3
1.6
3.1
+++
++
+
+++
+
+
+
+
4
08-TIVVV
4
08-AAAAA
4
4
08-HHHHH
08-WYHHH
4
4
08-WYHHHHH
08-GQWCSQY
1
Mutants are made of VpStyA2B as described in methods and were in analogy to the wildtype investigated.
The electron transfer yield (ETY) was calculated from the NADH-consumption vs. epoxidation rate. In case of
2
3
StyA1 an additional NADH:FAD oxidoreductase was needed (here RoStyBart) which could also assist StyA2B-like
4
proteins [6,24]. The plate screening was performed with clones expressing respective genes on an agar plate and
the indigo formation (+ little, ++ normal, and +++ significant indigo formed) was followed online by a camera.
n.a. = no activity measureable.
It is interesting that VpStyA2B is about 8.4-times more active on styrene than RoStyA2B [5,6].
The activity of the latter could be boosted by additional FAD-reductase, but, not for VpStyA2B.
Another difference between both StyA2B-proteins is the FAD-reductive power which is about 4.3-times
−
1
−1
higher in the case of VpStyA2B with a kcat of 22.3 s vs. that of RoStyA2B of 5.2 s . This higher
oxidoreductase efficiency of VpStyA2B might lead to the higher epoxidase activity, even, without an
additional FAD-reductase. Hence, the two domain protein VpStyA2B is a better biocatalyst as the
prototype RoStyA2B.
The mutants obtained were in analogy to the wildtype enzymes characterized for their capability
−
1
to convert styrene into styrene oxide. The wildtype VpStyA2B showed an activity of 159 mU mg
.
The VpStyA2B-variants were prepared similarly and assayed immediately in order to allow a direct
comparison. The following relative styrene epoxidase activities were determined: 84.9% for TIVVV,
163.5% for AAAAA and less than 2% in case of the other variants (HHHHH, WYHHH, WYHHHHH,
and GQWCSQY). This clearly shows the proposed linker region has significant effects on the epoxidase
activity of StyA2B-proteins.
From the nature of this proposed linker sequence it can be reasoned that smaller hydrophobic
residues yield higher activities compared to larger or more polar residues. This is indeed true for the
FAD-reductase as well as for the corresponding styrene epoxidase activity. And again the mutant
4
08-AAAAA showed a very promising catalytic behavior which was better than the wildtype in terms
of activity and efficiency (Tables 2 and 3). This means the electron transfer from NADH towards FAD
which yields reduced FAD and allows oxygen activation for catalysis was more efficient in case of
mutant protein AAAAA (an electron transfer yield of 3). In terms of epoxidase activity it was followed
by the TIVVV-mutant (an electron transfer yield of 4.4 at a lower epoxidation activity) which was even
more active with surplus of reduced FAD supplied by an additional FAD-reductase (Table 3; extra
RoStyBart). It achieved almost the same epoxidase activity as VpStyA1. This might allow to draw some
conclusions as it might be possible to generate more efficient and catalytic active self-sufficient two
domain proteins; here SMO-like monooxygenases. But, as the mutagenesis of the C-terminal part of the
StyA2-domain improved catalytic properties it might be possible to alter StyA1- or StyA-like proteins
at their C-terminal site in order to improve their catalytic properties. Furthermore, the generation of
artificial fusion proteins seems promising [20,29]. However, structural investigations are necessary in
order to elucidate the nature of substrate and cofactor binding.

Molecules 2018, 23, 809
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Various flavin-dependent monooxygenases accept sulfide-like compounds as substrates [
The latter can be converted to sulfoxides or sulfones in dependence of the employed biocatalyst [
4
–
1
11].
].
–
3
Therefore, the specific activity of VpStyA1/VpStyA2B on a model substrate, here benzyl methyl sulfide
(
BMS), was determined. Specific activities were determined as described above for styrene in order to
−
1
allow a comparison (Table 2). Maximum specific activity on BMS was determined to 3.113 U mg
VpStyA1 and 1.616 U mg VpStyA2B, respectively. This is about 25 to 26-times faster as the specific
−
1
activity on styrene. Hence, the two-component monooxygenase of strain EPS has a preference for this
sulfide. Also this high sulfoxidase activity is comparable to other SMOs [8,10], but the high selectivity
yielding almost pure (S)-enantiomers is somewhat outstanding.
2
.5. Biotransformation of Sulfides
For the biotransformation experiments, another fresh batch the enzyme (VpStyA1 and VpStyA2B)
was produced and partially purified. The enriched protein fractions obtained were sufficient
to study substrate range and conversion. Sulfoxides were chosen as target compounds since
VpStyA1/VpStyA2B showed an enhanced sulfoxidase activity. Respective sulfides were used as
substrates (Table 4). Both components converted the sulfides into corresponding sulfoxides. A further
oxidation to sulfones was not observed. And BMS was the best substrate. VpStyA1 produces almost
exclusively the (S)-sulfoxides and good conversions of up to 95% were achieved. VpStyA2B also
preferably produces the (S)-enantiomer but at a lower enantiomeric excess. The overall conversions
were much lower in case of the two domain protein. This is in congruence to the kinetic investigations
and indicates that VpStyA1 is the main monooxygenase of the two-component system.
Table 4. 2 h Biotransformation of sulfides and styrene. Conversions of 2 mM substrate were analyzed.
VpStyA1
Conversion (%)
VpStyA2B
Conversion (%)
Substrate
Product
ee (%)
ee (%)
PMS
43.6 ± 1.9
59.8 ± 0.4
38.5 ± 1.7
32.4 ± 4.7
98 (S)
6.3 ± 0.3
4.3 ± 0.7
4.4 ± 0.4
6 ± 0.7
64 (S)
4
F-PMS
99 (S)
99 (S)
99 (S)
84 (S)
96 (S)
96 (S)
4
Cl-PMS
4
Br-PMS
BMS
95 ± 0.5
9.9 ± 0.4
97 (S)
13 ± 0.9
0.5 ± 0.1
n.d. (S)
45 (S)
Styrene
98.2 (S)
Phenyl methyl sulfide (PMS; thioanisole), 4-fluoro phenyl methyl sulfide (4F-PMS), 4-chloro phenyl methyl sulfide
4Cl-PMS), 4-bromo phenyl methyl sulfide (4Br-PMS), benzyl methyl sulfide (BMS); n.d. = not detectable.
(

Molecules 2018, 23, 809
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Earlier reports on SMO-based sulfoxidations showed often a high activity but low
enantioselectivity [ 10]. This is now improved by means of VpStyA1, which shows excellent
1–
conversions of sulfides in short reaction times while also reaching a high ee-value of the respective
products (Table 4). It allows access to the (S)-enantiomers. It would be also interesting to get an
enzyme producing the (R)-enantiomers with a high ee. A monooxygenase from metagenome allows
one to produce (R)-enantiomers, but at rather low ee-values (60–92%) and after much longer reaction
times [10].
3
. Materials and Methods
3
.1. Synthesis of Sulfoxides
Commercial sulfides served as base to produce sulfoxides which were applied as standards for
analytical methods. The chemical sulfoxidation was achieved according to the protocol published
earlier [31] as it provided high yields of desired products. First a solution of 7 mmol sulfide in 100 mL
methanol and 20 mL water plus 10 mL titanium (III) chloride (16% aqueous solution) was prepared.
Then dropwise a hydrogen peroxide solution (3.2 mL 30% aqueous in 15 mL methanol) was added
◦
while constantly stirring at ambient temperature (about 20 C). Latest after 25 min the substrate was
completely converted and the reaction had been stopped by adding 50 mL water. Sulfoxides formed
were extracted by chloroform (three times) and subsequently dried over anhydrous magnesium sulfate.
Chloroform was further removed under reduced pressure. Success of reactions and purity of products
was determined as described elsewhere [32].
3
.2. Nucleotides, Sequence Analysis and Molecular Modelling
Sequence analyses based on a previously performed investigation [5]. Thus accession numbers
of the E2-type SMO components originating of Variovorax paradoxus EPS were already available:
ADU39063 (VpStyA1) and ADU39062 (VpStyA2B). These were used for a BLASTP search in order
to identify related or even homologous proteins. This allowed to generate an amino acid sequence
alignment in analogy to earlier reports [
of a dendrogram and the homology modelling. As templates for homology modelling the two
available structures (PDB StyA: 3IHM, and PDB PheA2: 1RZ0) were employed [19 33]. The C-terminal
5,21]. This served as a template for the subsequent calculation
,
StyA2-part was linked to the N-terminal StyB-part by a random loop which gets obvious from
Scheme 1 and Figure 1, respectively. The following tools were used: MEGA7 for the alignment,
Modeller program version 9.19 for the comparative homology modelling as well as GenDoc and PyMol
V1.1r1 for alignment and 3D-structure visualization [9,34–36].
3
.3. Bacterial Strains and Cultivation
Escherichia coli strains DH5 and BL21 (DE3) pLysS were cultivated for cloning and expression
as described elsewhere [ 37]. A list of genes, primers and plasmids for this work is presented in
α
5,6,9,
the Supplemental Information (Table S1). Indigo formation was observed during the cultivation and
gene expression experiments. A plate screening in order to monitor indigo formation was set up as
follows. The expression clones were transferred on a M9 mineral media agar-plate in a predefined
◦
grid, followed by a 22 h incubation at 30 C [38]. The solid media was supplemented with antibiotics
as described previously and 0.5 mM isopropyl-β-D-thiogalactopyranoside for induction of protein
expression [ 37]. Indigo formation was started by spraying the plates with an indole-substrate
5,6,9,
solution (5 Mm Tris-HCl, pH 7.5, 50 mM indole, 20% DMSO). The plates were then transferred onto a
white light-table in a darkened box equipped with a camera. Pictures of the plates were taken in a 30 s
interval. The color formation of each colony was monitored and normalized upon colony size to get a
time-resolved intensity profile for each clone.

Molecules 2018, 23, 809
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3
.4. Molecular Genetic Work
The DNA sequences of VpstyA1 (Accession number: MF781076) and VpstyA2B (MF781075) were
optimized for the codon usage and GC content of Acinetobacter baylyi ADP1 as described previously [ 39].
The genes were purchased in a pEX-A vector system from Eurofins MWG (Ebersberg, Germany)
9,
0
0
with 5 -NdeI and 3 -NotI restriction sites allowing for subcloning into pET16bP [9]. Site-directed
mutagenesis of the linker area was done by using the GeneMorph II EZClone Domain Mutagenesis
Kit (Agilent Technologies, Ratingen, Germany). A megaprimer was generated by amplifying the
target region with a primer that contains the desired mutation. This megaprimer was annealed to the
parental plasmid and extended in the EZClone reaction. Afterwards, the parental DNA was DpnI
digested and the remaining pET16bP construct harboring the mutation was transformed into E. coli
BL21 cells for gene expression. Successful mutation of the target genes was proven by sequencing of
the plasmid using the pET16-check-fw/pET16-check-rev primer [40].
3
.5. Protein Purification and Quantification
Recombinant protein production and purification was done as described previously [9,24]. Protein
concentration was determined by the Bradford method employing bovine serum albumin as a
standard [41]. The purity of protein preparations was controlled by SDS-PAGE as described earlier
for those types of proteins [
employed for RoStyA1 and RoStyA2B [
determined by RP-HPLC connected to a diode array detector for UV/Vis range.
5,
6]. Further, the FAD content of proteins was determined by the protocol
6
]. Therefore, authentic standards for FMN and FAD had been
3
.6. Enzyme Assays and Product Analysis
Flavin oxidoreductase activity was determined spectrophotometrically (Cary 50, Varian,
Agilent Technologies, Ratingen, Germany) by quantifying NAD(P)H consumption at 340 nm
− −1
nm = 6.22 mM cm ). One unit of enzyme activity is defined as the amount required to oxidize
40
1
(ε
3
1
µmol of NAD(P)H per min. All measurements were carried out in triplicate. The standard assay
(
1
1 mL) consisted of 20 mM Tris-HCl, pH 7.5, 68 µM flavin co-substrate (FAD, FMN or riboflavin) and
◦
77
µM NADH. After incubating the mixture for 10 min at 30 C, the reaction was started by adding
an appropriate amount of enzyme. For estimating steady-state specific activities, initial reaction rates
were determined using 4.1 to 90.2 M FAD and 7.9 to 164 M NADH. Specific activities were obtained
µ
µ
by nonlinear regression analysis applying KaleidaGraph 4.5 (Synergy Software, Reading, PA, USA),
assuming Michaelis-Menten kinetics.
Monooxygenase activity assays and standard HPLC analytics were performed as described
previously [5,9]. Reduced FAD was supplied by an additional reductase RoStyBart [24] or by chemical
reduction with 10 mM BNAH, respectively. The 2 h biotransformations were done according to the
standard monooxygenase assay by adding the respective sulfides instead of styrene as substrate.
Enantiomeric excess of obtained products were analyzed as described previously for epoxides [
sulfoxides [32].
5] and
4
. Conclusions
The two-component monooxygeanse VpStyA1/VpStyA2B originating of Variovorax paradoxus
EPS was herein described as the first proteobacterial E2-type representative. It also comprises a
two domain protein as an NADH:FAD-oxidoreductase (VpStyA2B). Both proteins were successfully
produced by recombinant gene expression. The enzyme converts styrene in an enantioselective manner
as well as several aryl alkyl sulfides. However, it was by far more active (3.3 to 22-times) on these
sulfides as on styrene. Therefore, this two-component enzyme can be proposed as an enantioselective
sulfoxidase. Furthermore, the potential linker region of the two domain StyA2B-like protein was
identified and mutated. It was demonstrated that this linker region has effects on the reductase (B) as
well as monooxygenase (A2) domain. Structural and kinetic studies may reveal the effect of the linker

Molecules 2018, 23, 809
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on the individual domains of VpStyA2B and related monooxygenase. Thus, this monooxygenase of
strain EPS is a suitable candidate for structural investigations as well as to develop an enantioselective
catalyst for sulfoxidation reactions.
Supplementary Materials: Supplementary materials are available online. Figure S1: Sensitivity of VpStyA2B
towards putative inhibitors determined by applying the NADH:FAD oxidoreductase assay, Figure S2: Flavin
determination of denatured VpStyA2B, Figure S3: SDS-PAGE analysis of protein preparations: VpStyA1 and
VpStyA2B, Table S1: Strains, plasmids and primers used in this study.
Acknowledgments: We thank the DECHEMA for support by means of a Max-Buchner-Research Fellowship to
Dirk Tischler (MBFSt 3339) and the Saxon Government for funding (SAB 100263733). We appreciate the support
and valuable discussions by R.W. and N.S. (University of Leipzig) during drafting the manuscript. We thank
Carolin Großmann for support with the protein determination. Support for the open access submission was
obtained from the Ruhr University Bochum.
Author Contributions: D.T. and T.H. conceived and designed the experiments; R.S. performed the mutagenesis
and analyzed mutants with respect to sequence and activity. S.R.K. performed the synthesis of sulfoxides
and contributed to the analysis; R.S., A.S., L.S., K.J. produced and analyzed the proteins, conducted the
biotransformations and analyzed the data; D.T. and T.H. wrote the paper. All authors approved the final
version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(