BBA - Proteins and Proteomics 1865 (2017) 1770–1780

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BBA - Proteins and Proteomics

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N-terminus determines activity and specificity of styrene monooxygenase MARK

reductases
Thomas Heinea,b,⁎, Anika Scholtisseka,b, Adrie H. Westphalb, Willem J.H. van Berkelb,
Dirk Tischlera,⁎⁎
a
Environmental Microbiology, Interdisciplinary Ecological Center, TU Bergakadmie Freiberg, Leipziger Straße 29, 09599 Freiberg, Germany
b
Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands

A R T I C L E I N F O A B S T R A C T

Keywords: Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of
Fusion protein styrene to (S)-styrene oxide. The FADH2 co-substrate of the epoxidase component (StyA) is supplied by an
Flavoprotein NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems.
Rhodococcus opacus One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120.
Protein-ligand interaction
The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that
Fluorescence spectroscopy
E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the ob-
served specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the
first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part
(A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN.
Pre-incubation with FAD yielded a turnover number at 30 °C of 133.9 ± 3.5 s− 1, one of the highest rates
observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence
analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is
shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their
activity and specificity.

1. Introduction and one, designated as E2-type, containing epoxidase-like proteins

Styrene monooxygenases (SMO; EC 1.14.14.11) form a unique
group (group E) of flavoprotein monooxygenases [1–3]. SMOs are two-
enzyme systems, consisting of an epoxidase (StyA) and a flavin re-
ductase (StyB; EC 1.5.1.36). StyA epoxidases are structurally related to
group A flavoprotein monooxygenases [1,4], while StyB reductases
have structural properties in common with iron reductases [5]. Being
involved in the microbial degradation of aromatic compounds in the
soil [6–9], SMOs catalyze the regio- and enantioselective epoxidation of
styrene into (S)-styrene oxide [10]. Others are supposed to be part of
indole detoxification [11]. Interestingly, all SMOs perform sulfoxida-
tion reactions [12–21], which is supposed to be a non-natural activity.
In recent years more and more SMOs were identified and described,
however, no clear nomenclature was introduced. We propose to define
two groups as suggested by Montersino et al. 2011: one with the most
common SMOs as E1-type proteins, homologous to PtStyA (epoxidase)
and PtStyB (reductase) from Pseudomonas taiwanensis VLB120 [22,23]
homologous to RoStyA1 and a fusion protein similar to RoStyA2B, both
from Rhodococcus opacus 1CP [13,24].
The SMO reaction starts with StyB, which generates FADH2, which
is then in turn used by the monooxygenase [23,25,26]. In StyA, FADH2
reacts with molecular oxygen to form flavin hydroperoxide, which
subsequently reacts with styrene to generate the enantiopure epoxide
[26,27]. After epoxidation, a flavin hydroxide remains, which subse-
quently eliminates water and is released as oxidized FAD. Although E1-
type StyBs are capable of reducing FAD, FMN, and riboflavin, epox-
idation of styrene by StyAs is only observed with FADH2 as co-sub-
strate.
So far, ten StyB reductases have been biochemically characterized
[21,23–26,28–31]. While being strictly NADH-dependent, they all are
homodimers and active with FAD, FMN and riboflavin. The N-termini
of the reductases vary in length, which is particularly true for the fusion
proteins (Fig. 1). In the latter, the reductases are linked to the mono-
oxygenase components by a shortened N-terminus, which is surprising,

⁎
Correspondence to: T. Heine, Environmental Microbiology, Interdisciplinary Ecological Center, TU Bergakadmie Freiberg, Leipziger Straße 29, 09599 Freiberg, Germany.
⁎⁎
Corresponding author.
E-mail addresses: thomas.heine@ioez.tu-freiberg.de (T. Heine), dirk-tischler@email.de (D. Tischler).

http://dx.doi.org/10.1016/j.bbapap.2017.09.004
Received 31 May 2017; Received in revised form 10 August 2017; Accepted 5 September 2017
Available online 06 September 2017
1570-9639/ © 2017 Elsevier B.V. All rights reserved.
T. Heine et al. BBA - Proteins and Proteomics 1865 (2017) 1770–1780

Fig. 1. Multiple sequence alignment of StyB reductases and related flavin:NADH oxidoreductases. The artificial RoStyBart serves as default. RoStyB Rhodococcus opacus 1CP (accession
number and/or PDB ID: AII82582), RoSty(A2)B Rhodococcus opacus 1CP (ACR43974), VpSty(A2)B Variovorax paradoxus EPS (ADU39062), PpStyB Pseudomonas putida S12 (4F07), PtStyB
Pseudomonas taiwanensis VLB120 (AAC23719), PpStyB Pseudomonas putida SN1 (ABB03728), AbStyB Acinetobacter baylyi ADP1 (YP_047251), R5StyB Rhodococcus sp. ST-5 (BAL04133),
R10StyB Rhodococcus sp. ST-10 (BAL04130), SgcE6 Streptomyces globisporus (AAL06698/4R82), PheA2 Geobacillus thermoglucosidasius A7 (AAF66547/1RZ1), NphA2 Rhodococcus sp.
strain PN1 (BAB86379), HpaC Thermus thermophilus HB8 (2ED4), TTHA0420 Thermus thermophilus HB8 (WP_011172509/1Y0A). Structural data are available for marked (*) sequences.
E2-type fusion proteins are truncated for the monooxygenase part. Shadings correspond to the level of conservation (black_100%; dark gray_80%; gray_60%). Scale under the alignment is
numbered according to the sequence of RoStyBart.

because structural studies of other flavin reductases illustrate that this
region is involved in substrate binding [32,33]. So far, only a single
crystal structure of a SMO reductase from Pseudomonas putida S12 is
known (PDB ID: 4F07; PpStyB S12). The first 13 amino acid residues of
this protein are not visible in the electron density map, and the func-
tional role of these residues in catalysis remains to be elucidated.
Preliminary investigations towards the substrate spectrum of E1-
type RoStyB pointed to a preference for FAD and inhibition with FMN
[21]. A substrate preference for FAD was also observed for HpaH C1
from Pseudomonas putida [34], pyrrole-2-carboxylate monooxygenase-
reductase from Rhodococcus sp. [35], TftC from Burkholderia cepacia
AC1100 [36], NphA2 from Rhodococcus sp. PN1 [37], PNP-B from
Bacillus sphaericus JS905 [38] and SgcEg from Streptomyces globisporus
[39], but an inhibition by FMN has never been reported. PheA2 from
Geobacillus thermoglucosidasius A7 is able to reduce FMN but shows no
tight interaction with this co-substrate in fluorescence titration ex-
periments [5,40].
The self-sufficient E2-type RoStyA2B contains fused epoxidase and
reductase domains, which function together as conventional SMOs
(StyA/StyB), but as a single polypeptide. RoStyA2B has a very low
flavin reductase activity [21,24,41], which could be due to the fusion of
the reductase to the much larger epoxidase. The N-terminus of the
RoStyA2B-reductase is about 12 to 22 residues shorter compared to
StyB reductases found in Pseudomonas, Variovorax and Rhodococcus

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T. Heine et al.
species [42] (Fig. 1). Most of the studied StyB reductases form homo-
dimers [2,21,23,26,29,30]. RoStyA2B forms oligomers, with a shift to
the dimeric form under reducing conditions. This suggests that both the
epoxidase and reductase in the fusion protein are dimers, constraining
the range of positions and conformations of the linkers between the two
enzymes [21]. StyB reductases usually follow a sequential mechanism
[23,25,30]. However, we could observe a shift to a double-displace-
ment reaction at higher FAD concentrations in an artificially con-
structed mimic of the fusion protein [31].
To obtain more insight in the function of the N-terminal part of the
RoStyB proteins, we deleted the monooxygenase part (A2) of RoStyA2B
and added the first 22 residues of RoStyB, resulting in artificial protein
RoStyBart. The redesigned reductase has been characterized with re-
spect to the parental oxidoreductases.
2. Materials and methods
2.1. Chemicals and enzymes
FAD, FMN and riboflavin were purchased from Sigma-Aldrich
(Steinheim, Germany) and Applichem (Darmstadt, Germany). NADH
was purchased from Gerbu (Heidelberg, Germany). Enzymes for cloning
purposes were received from MBI Fermentas (St. Leon-Rot, Germany)
and New England Biolabs GmbH (Frankfurt am Main, Germany).
2.2. Bacterial strains, plasmids and culture conditions
Escherichia coli strain DH5α and strain BL21 (DE3) pLysS were
cultivated for cloning and expression purposes as described elsewhere
[43]. Plasmids are listed in Table 1.
2.3. Multiple sequence alignment of StyB reductases and protein structure
modeling of RoStyBart
A multiple sequence alignment of StyB reductases was obtained
using MEGA version 6 [44]. Sequences were obtained from the NCBI
protein database (www.ncbi.nlm.nih.gov). The alignment served as
initiation point for comparative modeling. Here, PheA2 (PDB ID:
1RZ1), HpaC (2D36), CobR (4IRA) and a putative oxidoreductase from
Rickettsia felis (4L82) served as the structural templates [5,45–47] and
an iterative approach was used with Modeller Software 9.14 [48] to
compute a RoStyBart model-structure.
2.4. Construction of expression clones
The rostyBart gene (Accession number: MF124795) was purchased
from Eurofins MWG (Ebersberg) in a pEX-A vector system allowing for
ampicillin resistance selection. The DNA sequence was optimized for
the codon usage and GC content of Acinetobacter baylyi ADP1 with the
OPTIMIZER tool [21,49]. 5′-NdeI and 3′-NotI restriction sites were
added and used for subcloning into pET16bP to obtain the expression
construct pRoStyBart_P01, from which recombinant proteins can be
obtained as His10-tagged proteins.
Table 1
Plasmids used in this study.
Plasmid Relevant characteristic(s)
pET16bP pET16b with additional multicloning site; allows production of recombinant proteins with an N-terminal His10-tag
pEX-A-PfStyBart pfstyBart (524-bp NdeI/NotI fragment) cloned into pEX-A
pEX-A-RoStyBart rostyBart (557-bp NdeI/NotI fragment) cloned into pEX-A
pRoStyA2B_P01a rostyA2B of R. opacus 1CP (1722-bp NdeI/NotI fragment) cloned into pET16bP
pRoStyB_P01a rostyB of R. opacus 1CP (545-bp NdeI/NotI fragment) cloned into pET16bP
pPfStyBart_P01 pfstyBart (524-bp NdeI/NotI fragment) cloned into pET16bP
pRoStyBart_P01 rostyBart (557-bp NdeI/NotI fragment) cloned into pET16bP
a
The original designations of plasmids and genes were pSRoA2B_P01 and pSRoB_P01, respectively.
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BBA - Proteins and Proteomics 1865 (2017) 1770–1780
2.5. Gene expression, purification and storage of recombinant proteins
The expression screening was performed with different media in
100-mL Erlenmeyer flasks for all constructs, allowing for the potential
production of recombinant reductases, in analogy to earlier studies
[13,21]. Expression, purification and storage of RoStyA2B and RoStyB
were performed as described earlier [21,24].
For RoStyBart, expression took place in a 3-liter biofermenter. E. coli
BL21 (pRoStyBart_P01) was cultivated in LBNB medium [50]
(100 μg mL− 1 ampicillin and 50 μg mL− 1 chloramphenicol) at 30 °C
until an OD600 of 0.4 was reached. The high amount of salt and pre-
sence of betaine can prevent the formation of inclusion bodies. The
batch was subsequently cooled to 20 °C. Expression was induced at an
OD600 of 0.6 by adding 0.1 mM of isopropyl-β-D-thiogalactopyranoside
(IPTG), and growth was continued for 20 h at 20 °C (120 rpm). Cells
were harvested by centrifugation (5000 ×g, 30 min, 4 °C), resuspended
in 10 mM Tris-HCl buffer (pH 7.5) and stored at −80 °C.
RoStyBart was obtained as soluble protein. Crude extract was pre-
pared from freshly thawed biomass by disruption in a precooled French
Pressure cell, followed by centrifugation to remove cell debris
(50,000 × g, 2 h, 4 °C). The supernatant was applied to a 1-mL HisTrap
FF column pre-equilibrated in 10 mM Tris-HCl, 0.5 M NaCl, 25 mM
imidazole, pH 7.5 (loading buffer), using an ÄKTA fast-performance
liquid chromatography system (GE Healthcare). The column was wa-
shed with 10 column volumes of loading buffer to remove nonspecific
bound proteins. RoStyBart was eluted with a linear imidazole gradient
up to 500 mM over 30 column volumes. Fractions with NADH:FAD
oxidoreductase activity (see paragraph 2.7.) were pooled and con-
centrated using Sartorius Vivaspin 20 filters (5000 MWCO) at 4 °C. The
concentrate was passed through a 10 mL Econo-Pac 10DG desalting
gravity-flow column (Bio-Rad) to remove remaining imidazole and
sodium chloride. Protein obtained was kept in storage buffer (10 mM
Tris-HCl, 50% [v/v] glycerol, pH 7.5) at − 20 °C.
2.6. Protein determination, flavin content analysis and analytical gel
filtration
Recombinant proteins were subjected to discontinuous sodium do-
decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [43] in
order to determine purity and subunit molecular size. Protein con-
centration was determined with the Bradford method [51], using
BradfordUltra reagent (Expedeon) and bovine serum albumin (Sigma)
as reference protein.
The presence of non-covalently bound flavin in RoStyBart was
analyzed using RP-HPLC. For that, 50 μL of 70 μM purified protein in
10 mM Tris-HCl (pH 7.5) were heated at 95 °C for 5 min. After cen-
trifugation (16,000 ×g, 10 min, 4 °C) 5 μL of the supernatant were
applied on a Eurospher C18 column (250 mm length by 4 mm i.d., 5 μm
particle size, 100 Å pore size; Knauer, Germany). RP-HPLC was per-
formed with 50 mM sodium acetate (pH 5) using a linear gradient of
15% to 60% methanol at a flow of 0.7 mL min− 1 to elute the flavins.
5 μL of 50 μM of each flavin species (net retention volumes: 6.40 mL for
FAD, 7.42 mL for FMN and 9.60 mL for riboflavin) were applied as
Source or reference
U. Wehmeyer
MWG Eurofins
MWG Eurofins
[24]
[21]
This study
This study
T. Heine et al.
references. The detection wavelength was 450 nm.
Flavin content of recombinant proteins was measured spectro-
photometrically at a wavelength of 450 nm (assuming a molar ab-
sorption coefficient for protein-bound flavin, ε450 nm =
11.3 mM− 1 cm− 1) [52]. In addition, the flavin fluorescence was
measured with excitation at 450 nm and emission detected at 525 nm.
Analytical gel filtration of apo- and holo-form of RoStyBart was
performed on an ÄktaFPLC system (GE Healthcare) as described earlier
[21]. The holo-form of RoStyBart was analyzed under oxidized and
reduced conditions. Identity of the eluted proteins was checked by SDS-
PAGE and the standard activity assay.
2.7. Activity assay and inhibition studies
Flavin oxidoreductase activity was determined spectro-
photometrically (Cary 50, Varian) by quantifying NAD(P)H consump-
tion at 340 nm (ε340 nm = 6.22 mM− 1 cm− 1) [53]. One unit of en-
zyme activity is defined as the amount required to oxidize 1 μmol of
NAD(P)H per min. All measurements were carried out in triplicate. The
standard assay consisted of 20 mM Tris-HCl, pH 7.5, 60 μM flavin co-
substrate (FAD, FMN or riboflavin) and 175 μM NAD(P)H in 1.0 mL.
After incubating the mixture for 10 min at 30 °C, the reaction was
started by adding an appropriate amount of enzyme (around 70 nM).
For estimating steady-state kinetic parameters, initial reaction rates
were determined using 2.5 to 60 μM FAD and 10 to 175 μM NADH.
RoStyBart was applied either as apo- or as holo-enzyme where the
purified flavin-free enzyme was considered as apo-form and the enzyme
pre-incubated with FAD was designated as holo-form. For the latter, the
stock enzyme solution (7 μM) was incubated (10 min, room tempera-
ture) in 20 Tris-HCl, pH 7.5 with 60 μM FAD. Kinetic parameters were
obtained by nonlinear regression analysis applying KaleidaGraph 4.5
(Synergy Software), assuming Michaelis-Menten kinetics.
The pH preference for holo-RoStyBart was tested in an assay con-
taining 100 mM sodium phosphate buffer, 60 μM FAD and 175 μM
NADH, covering a pH-range from 5.4 to 8. In order to elucidate the
temperature optimum for the oxidoreductase activity of RoStyBart, the
holo-enzyme was applied to a pre-equilibrated standard assay (20 to
47 °C). For evaluation of the thermal stability, the holo-enzyme was
incubated for 10 min in a microcentrifuge tube equilibrated in 20 Tris-
HCl, pH 7.5 with 60 μM FAD to the respective temperature (20 to 52 °C;
Thermomixer, Eppendorf), and applied to the standard assay at 30 °C.
To evaluate the flavin specificity, RoStyBart (7 μM) was pre-in-
cubated at room temperature in 20 mM Tris-HCl, pH 7.5, containing
either 60 μM of FAD, FMN or riboflavin. After a period of 60 min, the
holo-protein obtained was applied to a standard assay containing either
60 μM of FAD, FMN, or riboflavin, or a FAD/FMN (1:1) mixture, re-
spectively. RoStyB was treated similarly and assayed for conversion of
FAD or FMN. In addition, oxidoreductase activity was measured, using
the standard assay, of samples taken periodically up to 60 min from an
incubation at 4 °C or 22 °C of RoStyBart (7 μM) added to 20 mM Tris-
HCl, pH 7.5, containing either 60 μM FAD or 60 μM FMN.
2.8. Spectrophotometric and fluorescence measurements
Flavin fluorescence titration was applied to determine the dis-
sociation constant (Kd) for the complex between RoStyB and FAD (Cary
Eclipse fluorescence spectrometer; Varian). RoStyB (3 μM) was titrated
with 0 to 10 μM FAD in 20 mM Tris-HCl, pH 7.5, and the FAD fluor-
escence signal was monitored at 525 nm upon excitation at 450 nm
(about 30 data points per titration step; standard deviation of < 1%).
The measurements were done at 13 °C in view of the low stability of
RoStyB.
All further spectroscopic measurements were carried out using a
Black Quartz Microplate (Hellma, clear bottom) and a SpectraMax M2e
and data were captured with the software SoftMax Pro (both Molecular
Devices, USA). Emission spectra were recorded for tryptophan
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BBA - Proteins and Proteomics 1865 (2017) 1770–1780
fluorescence (excitation wavelength = 280 nm, emission range
290–400 nm) and for FAD fluorescence (excitation wave-
length = 450 nm, cutoff wavelength = 455, emission ran-
ge = 476–650 nm). An equimolar amount of FAD was applied to apo-
protein to generate the holo-form of the proteins. Dissociation constants
of protein-ligand complexes were determined from tryptophan (ex-
citation wavelength = 280 nm, cutoff wavelength = 290 nm, emission
wavelength = 340 nm) and/or flavin (excitation wave-
length = 450 nm, cutoff wavelength = 515 nm, emission wave-
length = 525 nm) fluorescence titration experiments. NADH fluores-
cence was monitored at 460 nm (emission wavelength = 340 nm).
NAD+, NADH, FAD and FMN served as ligands for RoStyBart. Each
titration step was measured separately in a well at 13 °C covering a
range of 0 to 6 μM of the respective ligand. The enzyme was applied at a
concentration of 0.43 μM to 20 mM Tris-HCl, pH 7.5, in a final volume
of 200 μL. To prevent the influence of post flavin-binding events, each
set up was started by addition of enzyme and immediate data acquisi-
tion.
Dissociation constants of enzyme-flavin complexes were obtained
by using the nonlinear Generalized Reduced Gradient (GRG) algorithm
(Solver – Microsoft Excel) by fitting the fluorescence emission data (F)
to the model described by Eqs. (1) and (2).
F = fFAD ∗ [FAD]free + fComplex ∗ [Complex] + fEnzyme ∗ [Enzyme]free (1)
(K dapp + [FAD]total + [Enzyme]total )
1
− ((K dapp + [FAD]total + [Enzyme]total )2 − 4∗ [FAD]total ∗ [Enzyme]total ) 2
[Complex] =
2
(2)
Here, fFAD, fComplex and fEnzyme are the fluorescence conversion factors
for free FAD, formed complex and free enzyme, respectively.
The time course of flavin binding to RoStyBart was monitored at
25 °C over 107 min by recording tryptophan fluorescence emission at
340 nm in 20 mM Tris-HCl, pH 7.5. Flavin co-substrates were applied in
ten-fold excess over the enzyme. Data fitting and calculation of rate
constants was executed with KaleidaGraph 4.5 (Synergy Software) ap-
plying Eq. (3) for the binding of FAD and Eq. (4) for the binding of
FMN. Here, F is the total fluorescence resulting from S1 and S2, re-
presenting the fluorescence of different protein-flavin species that are
formed, and B, the fluorescence of the remaining assay components.
F = B + S1 ∗e(−t ∗ kapp1) − S2 ∗e(−t ∗ kapp2) (3)
F = B + S1 ∗e(−t ∗ kapp1) (4)
3. Results
3.1. Multiple sequence alignment of StyB reductases and structure modeling
A multiple sequence alignment of the selected StyB reductases was
computed with MEGA6 [44] (Fig. 1). Sequences were obtained using
RoStyA2B (ACR43974) and RoStyB (AKM21224) as seed sequences for
BLASTP searches [54] in the non-redundant protein database (NCBI).
All StyB primary structures are rather short in length (149 to 182 aa)
and share large portions of sequence similarity. The motifs responsible
for NAD and flavin binding [32,33,39,55–58] are conserved.
Based on the presented sequence alignment, a dimeric model of
RoStyBart was computed using the crystal structures of PheA2, HpaC,
CobR and a putative oxidoreductase as templates (Fig. 2). If present in
the template structures, the (co-)substrates NADH and FAD were in-
cluded in the modeling [5,26]. The single tryptophan (Trp70) of RoS-
tyBart was found to be close to the isoalloxazine ring of FAD (distance
about 4 Å) (Fig. 2A).
The multiple sequence alignment depicted in Fig. 1 contains next to
all characterized StyB reductases also related reductases. In the fol-
lowing, all amino acid positions are numbered according to the
T. Heine et al. BBA - Proteins and Proteomics 1865 (2017) 1770–1780

Fig. 2. Surface map (A) and ribbon diagram (B) of the 3D-structural model of RoStyBart with bound FAD and NADH. Flavins are placed according to structural data of PheA2 (PDB ID:
1RZ1; bright orange) and PpStyB S12 (4F07; orange). NADH is positioned according to PheA2 (1RZ1). Protein backbones of the dimers are shaded in green or gray and the N-terminal
residues are shaded in red. In (A), residues that are proposed to be involved in (co-)substrate binding are shaded in orange (FAD), blue (NADH) and a flexible loop (aa 106 to 114) above
the FAD binding site (yellow), respectively. The only tryptophan within the RoStyBart sequence (Trp70) is close to the isoalloxazine ring (about 3.4 to 4 Å; magenta). Arg28 is part of the
N-terminal region and known to form hydrogen bonds towards NADH (indicated by dashed lines). For clarity, only one FAD and one NADH molecule is indicated in the dimer (B). (For
interpretation of the references to color in this figure legend, the reader is referred to the online version of this chapter.)

sequence of RoStyBart. The residues that are required for (co-)substrate
binding are highly conserved. The TANx3Sx10S, [STC]xxPP and GDH
motifs are localized at the active site or close to it [39,55,56]. Thr54,
Ala55, Asn56 and Ser/Cys71 are involved in binding the isoalloxazine
ring of the FAD [39,56–58]. Val38, Ala55, Ser105 and Phe167 form a
hydrophobic pocket to bind the dimethylbenzene moiety of the iso-
alloxazine ring [5,32,58]. Whereas the location of the FMN moiety is
similar, the AMP moiety of FAD can bind differently. In PheA2, the
adenine and ribose moieties interact with valine and asparagine re-
sidues (Lys50 and Gln103 in RoStyBart) at a small cleft on the surface of
each monomer [5,32]. Interestingly, the lysine residue replacing the
non-polar valine is only found in StyA2B-like SMOs and AbStyB (Aci-
netobacter baylii ADP1), which might affect the recognition of the AMP
part of FAD [55]. Pro77 and Phe104 may also form hydrogen bonds
with the adenosine moiety of FAD [58]. In PpStyB S12, the adenosine
moiety occupies the active site of an adjacent monomer [26]. This
might be due to a more open conformation of a loop that corresponds to
residues 106–114 in the RoStyBart model (Fig. 2A). This region is
variable in StyB reductases and thus no suggestion can be made how the
AMP part of FAD will bind in RoStyBart.
There is only one StyB structure available in the database [26]. In
this structure, the N-terminal residues 1–13 are not visible due to poor
electron density [26]. This suggests a high flexibility of the N-terminus.
As this region is close to the FAD/NADH binding pocket, it could in-
fluence the catalytic properties of the enzyme. Furthermore, the flexible
N-terminus might also be involved in the formation and transient sta-
bilization of a StyA-StyB complex [26].
3.2. Purification of RoStyBart and analytical gel filtration
RoStyBart was purified by metal affinity chromatography. SDS-
PAGE yielded a single band at a size of approximately 21 kDa (Fig. S1),
in accordance with the theoretical subunit mass of 21.8 kDa (including
the N-terminal His10-tag). HPLC analysis of the heat-treated RoStyBart
preparation showed no peaks at the expected positions of FAD, FMN or
riboflavin (Fig. S2), thus, the protein is purified mainly in its apo-form.
This is supported by the absorption and fluorescence properties of the
purified RoStyBart enzyme. Neither the characteristic flavin absorption
spectrum nor an emission signal at 525 nm after excitation at 450 nm
was observed.
The hydrodynamic properties of the SMOs from R. opacus 1CP were
studied by Riedel et al. [21]. The RoStyB apo-protein mainly occurs as
dimer. The apo- and holo-forms of RoStyA2B exist as higher-order
quarternary complexes in equilibrium with the dimer. However, under
reduced conditions there is a shift to the dimeric state. In this study, we
performed analytical gel filtration under similar conditions for RoSty-
Bart (Fig. S3). The apo-form of RoStyBart mainly occurs as a monomer.
If incubated with FAD, a clear shift to the dimeric state was detected for
the holo-form of RoStyBart, with minor formation of higher-order
structures. Under reduced conditions, the holo-enzyme almost solely
exists as a dimer. If incubated with FMN, there is less tendency to di-
merization. Under oxidized conditions, monomer and dimer of FMN-
bound RoStyBart are in equilibrium. Under reduced conditions, there is
a shift to the monomeric-state.
3.3. Catalytic properties of RoStyB, RoStyA2B and RoStyBart
3.3.1. Enzyme activity with FAD
The enzymes used here are not active with NADPH, thus, all oxi-
doreductase activities were assayed using NADH. The steady-state ki-
netic properties of RoStyA2B and RoStyB with FAD as co-substrate have
been reported before [21,24] and are summarized in Table 2. Worthy of
note is that pre-incubation of RoStyB with FAD does not change the
enzyme activity.
RoStyBart is very active with FAD (Fig. 3). However, the Michaelis
constants for FAD and NADH of this redesigned enzyme are quite dif-
ferent from that of RoStyB and RoStyA2B (Table 2). The KM, FAD values of
RoStyBart determined with apo-protein (KM,− FAD = 15.5 ± 1.2 μM), or
with apo-protein pre-incubated with FAD (KM,+FAD = 19.1 ± 1.2 μM)
are in the same range, as is the case for the KM values for NADH
(KM,NADH − FAD = 110.5 ± 9.9 μM; KM,NADH + FAD = 74.6 ± 4.4 μM).
Pre-incubation with FAD gives a three-fold increase in activity of RoS-
tyBartholo (kcat = 133.9 ± 3.5 s− 1) compared to RoStyBartapo
(kcat = 46.1 ± 2.1 s− 1) (Fig. 3, Table 2).
Upon incubation at 20 °C, RoStyBartholo is stable for at least 60 min
(Fig. S4). In a series of experiments where the standard activity of
RoStyBartholo was measured at various temperatures, a maximum was
found at around 40 °C. If RoStyBartholo is pre-incubated at various
temperatures for 10 min, the activity (measured at 30 °C) starts to drop
above 20 °C, an indication that the enzyme is not very thermotolerant
(Fig. S5). The pH optimum for RoStyBartholo lies in a range of 5.8 to 6.2
(Fig. S5).

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T. Heine et al. BBA - Proteins and Proteomics 1865 (2017) 1770–1780

Table 2
Kinetic parameters of RoStyBart compared to its templates and ancestors.

Enzyme Electron donor Electron acceptor Km kcat kcatKm− 1 Reference

μM μM μM s− 1 s− 1 μM− 1

RoStyBartapo NADH (10–175) FAD (60) 110.5 ± 9.9 46.1 ± 2.1 0.417 This study
NADH (175) FAD (2.5–70) 15.5 ± 1.2 34.3 ± 0.9 2.216
RoStyBartholo NADH (10–175) FAD (60) 74.6 ± 4.4 133.9 ± 3.5 1.795 This study
NADH (175) FAD (2.5–70) 19.1 ± 1.2 122.7 ± 2.8 6.419
RoStyA2B NADH (17.5–175) FAD (50) 58.0 ± 9.0 3.9 ± 0.3 0.068 [24]
(R. opacus 1CP) NADH (175) FAD (4–60) 26.0 ± 2.0 5.2 ± 0.2 0.203
NADH (175) FMN (4–60) 67.0 ± 22.0 4.8 ± 1.0 0.071
NADH (175) Riboflavin (4–60) 23.0 ± 2.0 3.0 ± 0.1 0.131
RoStyB NADH (2.5–300) FAD (200) 59.3 ± 4.2 26.9 ± 0.7 0.454 [21]
(R. opacus 1CP) NADH (250) FAD (5–300) 108.3 ± 18.3 32.9 ± 2.4 0.304

(See Supporting Table S1 for kinetic data of other StyB's).

The specific activity of RoStyBartholo depends on the enzyme con-
centration (Fig. 3C). A significant decrease was observed at con-
centrations below 0.75 μg mL− 1. When, upon dilution in the assay
mixture, dissociation of dimers occurs, the decrease in activity can be
explained if the formed monomers are not active. A dissociation con-
stant for the dimer of about 1 nM would describe the observed curve. As
described in paragraph 3.2, dimerization of RoStyBart is stimulated by
flavin binding (cf. Fig. S3).
3.3.2. Enzyme activity with different flavin cofactors
Interesting differences between RoStyB and RoStyBart are seen in
activity measurements started with apo-protein and FAD or FMN in the
assay solution (Fig. 4). RoStyB shows only about 10% activity with FMN
compared to the activity with FAD (Fig. 4A). Furthermore, the activity
with FAD and FMN is hardly dependent on the pre-incubation with
FAD. However, if RoStyBapo is pre-incubated with FMN, little activity is
found in the reaction with FAD and there is almost no activity with
FMN.
With RoStyBart, the activity with FAD and FMN is strongly stimu-
lated by pre-incubation with FAD (Fig. 4B). This suggests that FAD
serves as activity enhancing prosthetic group. Both RoStyBartapo and
RoStyBartholo are relatively more active with FMN than the corre-
sponding RoStyB proteins (Fig. 4). However, when RoStyBart is pre-
incubated with FMN, the enzyme is hardly active with both FAD and
FMN, suggesting that FMN functions as an inactive prosthetic group.
If riboflavin is used as flavin co-substrate for RoStyBartapo no

Fig. 3. Steady-state kinetic analysis of FAD:NADH oxidor-

eductase activity of RoStyBart. Specific activities are
plotted as function of FAD (A) or NADH (B) concentration
while keeping the amount of the other substrate constant.
Kinetic parameters were calculated using KaleidaGraph 4.5
(Synergy) assuming Michaelis-Menten kinetics. The stan-
dard assays were started with either RoStyBartapo (■; da-
shed line) or RoStyBartholo (●; solid line). (C) Specific ac-
tivity of RoStyBartholo as a function of enzyme
concentration. All measurements were done in triplicate.

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T. Heine et al. BBA - Proteins and Proteomics 1865 (2017) 1770–1780

Fig. 4. Relative activities of RoStyB (A) and RoStyBart (B)

with different flavin co-substrates. Enzymes were used as
apo-protein or pre-incubated for 60 min in a 60 μM flavin
containing solution and subsequently applied to the stan-
dard assay containing NADH and the respective flavin. All
measurements were carried out in triplicate. Results are
depicted relative to the respective holo-enzyme.

oxidoreductase activity is observed. Pre-incubation with riboflavin

neither yields activity with FAD, FMN or riboflavin as substrate. This is
again an indication that only FAD serves as activity enhancing pros-
thetic group of RoStyBart.

3.3.3. Kinetic mechanism of RoStyBart

In order to elucidate the kinetic mechanisms of the apo- and holo-
forms of RoStyBart, initial rates were determined over a range of
FAD:NADH ratios and depicted in double reciprocal plots (Fig. S6). In
case of RoStyBartapo, for NADH as well as for FAD as fixed (co-)sub-
strate, the different slopes intersect to the left of the ordinate (Fig. S6A
and Fig. S6B). In the first case, the lines intersect at the X-axis, in-
dicating a constant KM for FAD and a sequential mechanism. In the
latter case, the intersection falls into the second quadrant, which is
typical for a ternary complex comprising mechanism. The intersections
with the X-axis imply a decrease in Km for NADH upon increasing FAD
concentration. Thus, it can be assumed that FAD serves as the leading
substrate. At this stage the observed intersections suggest that
RoStyBartapo obeys a sequential ordered bi-bi mechanism. Further, the
proposed initial binding of FAD fits to a proposed role as a prosthetic
group. This is in accordance with mechanistic studies on RoStyBartholo
(Fig. S6C and Fig. S6D). For NADH as well as for FAD as fixed co-
substrate, parallel slopes are observed, which indicates a switch to a
ping-pong bi-bi mechanism compared to the apo-form.

3.4. Protein-flavin interaction

The binding of FAD to RoStyA2B, RoStyB and RoStyBart was de-
termined by monitoring flavin and/or tryptophan fluorescence emis-
sion. The FAD-fluorescence of RoStyBartholo is almost identical to that
of free FAD. In RoStyA2Bholo and RoStyBholo, the FAD-fluorescence is
about doubled or raised about 50% compared to that of free FAD, re-
spectively (Fig. 5A).
RoStyA2B and RoStyBart share a single tryptophan at position 49
and 70, respectively (Fig. 2). RoStyB, on the other hand, contains two
tryptophans at position 86 and 111 (Fig. 2). The maxima of the tryp-
tophan emission of RoStyBartapo and RoStyBartholo around 330 nm are
blue-shifted compared to that of the apo- and holo-forms of RoStyB and
RoStyA2B. This suggests that the tryptophan in RoStyBart is located in a
rather apolar environment which was confirmed by mapping the hy-
drophobicity onto the model structure (Fig. S8B). Upon FAD binding,
the tryptophan fluorescence of RoStyBartholo decreases by about 50%,
that of RoStyB by about 30%, and that of RoStyA2Bholo by about 70%,
compared to the apo-form (Fig. 5B). The Tryptophan is close to the
isoalloxazine ring (about 3.4 to 4 Å). Likely, in the holo-form of the
proteins, Förster resonance energy transfer takes place (FRET) from the
Fig. 5. (A) Fluorescence emission spectra of FAD (excitation wavelength = 450 nm) for
RoStyBartholo (orange), RoStyA2Bholo (blue) and RoStyB (green) normalized to the
emission maximum of free FAD (black). (B) Fluorescence emission spectra from trypto-
phans of RoStyBart (orange), RoStyA2B (blue) and RoStyB (green), whereby apo-protein
is shown by dashed lines and holo-protein by solid lines (excitation = 280 nm). Curves
were measured in duplicates. Assays contained 50 mM Tris-HCl (pH 7.5), 17.5 μM FAD
and 17.25 μM of the respective protein.
tryptophan to the FAD, thereby diminishing the tryptophan emission.
Tryptophan fluorescence studies revealed different kinetics for FAD
and FMN binding to RoStyBartapo. In case of incubation with FMN there
is a slow decrease in tryptophan fluorescence over time with a rate
constant kapp1 of 0.0233 ± 0.0003 min− 1 (Fig. 6A). For FAD, initially
a relatively fast rise (kapp2 = 0.448 ± 0.02 min− 1) is followed by a
slow decrease in tryptophan fluorescence (Fig. 6A). The slow step has
an apparent rate constant similar to that of binding of FMN
(kapp1 = 0.0206 ± 0.0003 min− 1).
The dissociation constants of the enzyme-FAD complexes of
RoStyBapo and RoStyBartapo were determined by fluorescence titration

1776
T. Heine et al.
experiments. To prevent the influence of post ligand-binding events,
each set up was started by addition of enzyme to the FAD solution and
immediate data acquisition (Fig. 6B). Clear binding of FAD was ob-
served for RoStyBapo and RoStyBartapo. From the tryptophan and flavin
fluorescence titration curves and global fitting, a Kd of 4.3 ± 0.2 μM
was estimated for the RoStyBapo-FAD complex assuming 1:1 binding.
This is in congruence with previous results that indicate weak binding
of FAD [21]. For RoStyBart, similar tryptophan and FAD titration ex-
periments resulted in a Kd of 0.65 ± 0.06 μM for the RoStyBartapo-FAD
complex. This value is almost one order of magnitude lower than the
dissociation constant determined for the RoStyA2Bapo-FAD complex
(Kd = 5.05 ± 0.52 μM; [13]).
4. Discussion
SMOs are playing an important role in the detoxification of styrene
in aerobic microorganisms. Rhodococcus opacus 1CP uses two SMO
types, which we here define as E1- and E2-type. E1-type proteins are
homologous to the PtStyA (epoxidase) and PtStyB (reductase) from
Pseudomonas taiwanensis VLB120 [22,23] and are usually embedded in
a styrene degradation cluster. E2-type SMOs contain epoxidase-like
proteins homologous to RoStyA1 and flavin reductases similar to the
reductase part of RoStyA2B, both from Rhodococcus opacus 1CP [13,24].
The E2-type is usually not part of a styrene degradation cluster [21].
Recent studies indicate that this second type might be involved in in-
dole detoxification [11]. This was earlier hypothesized [42] and a
convergent evolution of these SMO types was concluded [30]. The
phylogenetic analysis (Fig. 1 and Fig. S7) presented here reinforce this
hypothesis. The motif TANx3Sx10S discriminates E1- or E2-type StyB
reductases. The alanine residue of this motif varies towards E1-type
reductases that have valine or isoleucine at this position. In most E2-
type reductases, the second serine of the motif is altered to cysteine.
This region is supposed to be involved in binding the isoalloxazine ring
of the FAD [39,56–58]. The amino acid changes in the TANx3Sx10S
motif should not have large effects due to their chemical similarity.
Furthermore, only in case of E1-type StyB reductases the GDH motif is
altered to histidine or asparagine at the glycine position. In the RoS-
tyBart model, this glycine residue is close to the SxxPP motif of the
adjacent monomer and this change might affect dimer formation.
Several reductases of two-component flavoprotein monooxygenase
systems display flavin promiscuity while the oxygenase components can
utilize only FAD. Indeed, this behavior was reported for PheA1/PheA2
[40], RebH/RebF [59] and HpaA/HpaC [60]. On the other hand, there
are also two-component flavoprotein monooxygenase systems that
possess FAD specificity within the oxygenase and the reductase: SgcC/
SgcE6 [61], NphA1/NphA2 [37,62], TftD/TftC [63], HPAH C2/C1
[34], 4-NP monooxygenase [38] and pyrrole-2-carboxylate mono-
oxygenase [35]. In this context, it is important to note that RoStyB is the
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BBA - Proteins and Proteomics 1865 (2017) 1770–1780
Fig. 6. Time dependent change in the tryptophan fluores-
cence intensity of RoStyBart (A) due to the binding of a
flavin co-substrate (excitation at 280 nm; emission at
340 nm). Solid dots refer to FAD and blank dots refer to
FMN. The curves were fitted with Eq. (3) (for FAD; dashed
line) and Eq. (4) (for FMN; solid line). For RoStyBart, one
rate constant kapp1 = 0.0233 ± 0.0003 min− 1 was ob-
tained for the binding of FMN whereas the fitting of the
FAD trace gives a fast component
kapp2 = 0.448 ± 0.02 min− 1 and a slow component
kapp1 = 0.0206 ± 0.0003 min− 1. Every curve was at least
measured twice. (B) Relative fluorescence emission from
tryptophan of RoStyBart (0.43 μM) measured at 340 nm (●
solid line; excitation 290 nm) and FAD measured at 525 nm
(○ dashed line; excitation 450 nm) during titration with
FAD to estimate the dissociation constant of the apo-RoS-
tyBart-FAD complex. Binding of FAD was monitored in case
of RoStyB (3 μM) at 525 nm (□ dashed line; excitation
450 nm).
first SMO reductase, which is strictly FAD-dependent. In contrast to its
family members, FMN is an inhibitor instead of a substrate for RoStyB.
Thus, it can mark an evolutionary event for this enzyme class. All other
StyB reductases are not strictly FAD-dependent and thus may yield
unproductive side-activities.
In this study, we replaced the N-terminal region of RoStyA2B to get
further insight into the molecular determinants of the flavin specificity
of the SMO reductases. The RoStyBart enzyme was predominantly
purified as apo-protein (Fig. S2). This is somewhat surprising in view of
the proposed function of FAD as prosthetic group (vide infra).
Nevertheless, RoStyBart appeared to be one of the most active StyB
enzymes reported today (Fig. 7 and Table 2). Pre-incubation of RoS-
tyBart with FAD led to a strong increase in oxidoreductase activity
(Fig. 3). This activation appeared to be dependent on protein con-
centration and temperature (Fig. S4). In accordance with that, we could
observe an initial increase in tryptophan fluorescence followed by a
slow decrease over time (Fig. 6A). The need for higher concentrations
and temperatures may be due to the need for a dimeric structure with a
hydrophobic interface (vide infra). The optimal temperature for the
activation is located around 20 °C (Fig. S4 and S5A). A temperature
dependent activation towards the binding of FAD has been described
before for other dimeric flavoproteins such as D-amino acid oxidase
[64] and lipoamide dehydrogenase [65,66].
The activity of RoStyBart is difficult to compare with other SMO
reductases as the specific activity depends on the protein concentration.
PtStyB and PpStyB S12 were shown to reach activities of up to
700 U mg− 1 upon dilution [23,25,26] (Fig. 7). Interestingly, RoStyBart
shows an opposite behavior (Fig. 3C). The specific activity of this
Rhodococcus enzyme decreases when the protein is diluted. RoSty-
Bartholo occurs manly in the dimeric state whereas the apo-form tends
to form monomers and oligomers (Fig. S3). This supports that SMO
reductases are active as homodimers. The quaternary structure may
also be the reason for the remarkable higher activity of the artificial
construct compared to RoStyA2B. As described earlier [21] RoStyA2B
has two dimer interfaces. Possibly, there is a steric hindrance due to the
large oxygenase part that impedes a correct dimer formation and thus, a
high catalytic activity.
Intriguingly, the affinity of FAD for RoStyBart (Kd =
0.65 ± 0.06 μM) is strongly improved compared to that of RoStyA2B
(Kd = 5.05 ± 0.52 μM) and RoStyB (Kd = 4.31 ± 0.18 μM) (Fig. 6B).
The affinity of RoStyBart for FAD is slightly better than found with other
SMO reductases: PpStyB S12 (1.15 ± 0.14 μM), PtStyB (2.3 ± 0.3 μM)
and AbStyB (1.82 ± 0.02 μM) [23,26,30]. A dissociation constant in the
micromolar range is common for NADH-flavin reductases [60,67–69]
whereas few also bind the FAD with nanomolar affinity [5,70].
Several flavin binding modes are feasible as shown for related re-
ductases. In PheA2, a cofactor FAD binds tightly with the isoalloxazine
ring directed to the dimer interface and the adenosine moiety stabilized
T. Heine et al.
Fig. 7. Comparison of the highest determined activities (A) for SMO reductases while using FAD and NADH as (co)-substrates. The relative catalytic efficiencies for FAD are shown in (B)
[13,21,23,24,28–30]. AbStyB was assayed at 25 °C and PpStyB SN1 at 37 °C.
by a loop after the third α-helix (which can also be found in the
RoStyBart model). PheA2 acts according to a ping-pong bi-bi me-
chanism. After reduction of the FAD cofactor by NADH and subsequent
release of NAD+, a flavin substrate binds at the NADH-binding site and
takes the electrons from the reduced FAD cofactor [5]. In contrast,
PpStyB S12 does not use a tightly bound FAD cofactor, which might be
due to a more flexible loop compared to PheA2. Therefore, the ADP
moiety of FAD can occupy the NADH binding site of an adjacent
monomer (Fig. S8A) [26].
A peculiar feature of RoStyB is the inhibition by FMN and riboflavin.
This is the first report about such a behavior in NADH:flavin oxidor-
eductases. Moreover, this property remained in the artificial construct
RoStyBart. Fluorescence kinetic data support a difference in binding
mode between FAD and FMN (Fig. 7). For incubation with FAD, the fast
component (kapp2 of 0.448 ± 0.02 min− 1) might reflect binding as a
prosthetic group to the flavin binding site. This is in accordance with
activation of RoStyBart during incubation with FAD (Fig. S4) which
happens in a similar time range. Further, RoStyBartholo shows a ping-
pong bi-bi mechanism, implying a second binding site for a flavin co-
substrate. Binding of FAD to that site might be reflected by the slow
decrease in fluorescence (kapp1 of 0.0233 ± 0.0003 min− 1). In con-
trast, when RoStyBart is incubated with FMN, the fast component is
missing and only the slow component can be determined. Thus, it can
be suggested that FMN does not bind to the flavin binding site as a
prosthetic group. Further, the rate of this slow decrease in fluorescence
(kapp1 of 0.0206 ± 0.0003 min− 1) is in a similar range when com-
pared to the binding of FAD to the co-substrate site as well as to the
inactivation of the enzyme (ki, app of 0.046 ± 0.001 min− 1, Fig. S4). It
is likely, that the co-substrate flavin has to bind in a closed conforma-
tion as with NADH (Fig. 2). As this is not possible for FMN, it will
occupy the second (co-substrate) binding site leading to inhibition of
the enzyme. This state might be stabilized by Arg28 (Fig. S8A). This is
supported by analytical gel-filtration experiments as RoStyBart showed
less dimer formation if FMN is applied under oxidized conditions and
dissociates to the monomeric state under reduced conditions (Fig. S3B).
This implies that FMN binds preferably to the NADH-binding site
without stimulating dimer formation. As only the N-terminal part is
different compared to RoStyA2B, this region seems to impact binding of
the flavin to the NADH binding site and in case of FMN binding is
unfavorable for catalysis.
5. Conclusion
RoStyB from Rhodococcus opacus 1CP is the first NAD(P)H-depen-
dent flavin reductase that is highly specific for FAD. RoStyA2B, a self-
1778
BBA - Proteins and Proteomics 1865 (2017) 1770–1780
sufficient SMO originating from the same strain, is less active as RoStyB,
but can reduce various flavins [24]. In this study we created an artificial
construct designated RoStyBart by joining the N-terminus of RoStyB
with the reductase part of RoStyA2B. The resulting RoStyBart combines
the flavin specificity of RoStyB with a higher activity and affinity for
FAD. This demonstrates for the first time that the NADH:FAD oxidor-
eductase activity of an natural E2-type SMO is affected by the N-
terminal fusion to the epoxidase subunit.
The kinetic studies of RoStyBartapo indicate FAD as leading substrate
in a sequential mechanism. The fluorescence investigations revealed a
fast step upon FAD binding, which does not occur in case of FMN
binding. This suggests that the AMP moiety of FAD is needed to trigger
RoStyBart in order to allow proper NADH binding and oxidation. A
second flavin binding site can be assumed as RoStyBartholo follows a
ping- pong mechanism. Thus, it is likely that a co-substrate FAD binds
to the NADH-binding site. FMN is supposed to block this site and in-
hibits the enzyme. Structural studies and further engineering of the N-
terminal region might help understanding the reasons for the change or
limitation in the co-substrate specificity.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgements
The authors thank for the funding by the European Social Fund and
the Saxon Government (GETGEOWEB: 100101363).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbapap.2017.09.004.
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