Fus‐SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency

The styrene monooxygenase, a two‐component enzymatic system for styrene epoxidation, was characterised through the study of Fus‐SMO – a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus‐SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well‐distanced arrangement (45–50 Å) facilitated by the flexible linker‘s loopy structure. Pre‐steady‐state kinetics elucidated the FADox reduction intricacies (kred=110 s−1 for bound FADox), identifying free FADox binding as the rate‐determining step. The aerobic oxidation of FADH2 (kox=90 s−1) and subsequent decomposition to FADox and H2O2 demonstrated StyA′s protective effect on the bound hydroperoxoflavin (kdec=0.2 s−1) compared to free cofactor (kdec=1.8 s−1). At varied styrene concentrations, kox for FADH2 ranged from 80 to 120 s−1. Studies on NADH consumption vs. styrene epoxidation revealed Fus‐SMO′s ability to achieve quantitative coupling efficiency in solution, surpassing natural two‐component SMOs. The results suggest that Fus‐SMO exhibits enhanced FADH2 channelling between subunits. This work contributes to comprehending FADH2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.


Introduction
The flavin-dependent styrene monooxygenase (SMO) is a twocomponent enzymatic system that belongs to the group E of the monooxygenases (GEMs). [1]In the past, all GEMs were annotated as SMOs that were further divided in two sub-types (E1 and E2). SMO naturally catalyses the asymmetric epoxidation of styrene and derivatives thereof, by consuming molecular oxygen and NADH (Figure 1). StyB is the first component in the catalytic mechanism and catalyses the reduction of flavin adenine dinucleotide (FAD ox ) at the expense of NADH.After this step, the reduced cofactor (FAD hq ) must be transferred to the second (reelaborated from ref. [4] c) the flavin cycle within the catalytic mechanism of SMO.component, namely StyA, which catalyses the reaction between molecular oxygen and FAD hq to form the flavin hydroperoxide (FAD OOH ).This FAD OOH formed in StyA's active site reacts with the bound substrate (e.g. styrene) to generate enantiomerically pure styrene oxide.The flavin-4a-hydroxide splits off water and oxidised flavin is regenerated, which can enter another catalytic cycle.
Kinetic analysis of the FAD ox reduction catalysed by StyB showed that the reaction proceeds through a sequential binding mode in which NADH binds before FAD ox to form a ternary complex StyB-FAD ox -NADH. [5]5b] Other authors working with manmade SMO enzyme fusions have postulated a sequential mechanism at low FAD concentration and a double-displacement mechanism at high FAD concentration. [6]tyB was crystallised as a homodimer (PDB 4F07; MM of 18.4 kDa for each protomer) in which each protomer participates and shares the binding of two molecules of FAD ox .5b] StyB recombinantly expressed in E. coli is completely obtained as inclusion bodies, thereby requiring in vitro refolding before any biochemical characterisation. [5]5a] In contrast to StyB, StyA is commonly well-produced in E. coli in soluble form.
Kinetic parameters for the catalytic cycle of the SMO have been determined over the past two decades (Scheme 1).Two research groups independently reported the dissociation constant (K D ) for FAD ox from StyB to be 1.2 μM or 2 μM, and the rate for the reduction of FAD ox to FAD red (i.e. FADH 2 ) to be 49 s À 1 or 60 s À 1 (independent data obtained at different StyB, FAD ox and NADH concentrations, and at 15-30 °C). [5]The affinity of NADH for StyB was also in the micromolar range (K M = 20 μM).3a,5a] These data show that StyB has a ca.1000 times higher tendency to bind FAD ox than StyA, which can be attributed to an evolutionary advantage since FAD ox is the substrate of StyB and FAD hq is the substrate of StyA.Additionally, the K D for FAD hq is ca.12 times higher for StyA than StyB that may aid for FAD hq transfer during the catalytic cycle.
Regarding the efficiency of the bi-component StyA-StyB system, previous studies showed that efficient coupling between reduction of FAD ox catalysed by StyB and styrene hydroxylation catalysed by StyA is obtained when the total free flavin concentration (i.e. FAD ox plus FAD hq ) is kept low in solution while StyA is in hundred times or more molar excess than StyB. [8]The low concentration of total free flavin in solution also prevents the occurrence of the chemical-side reaction that practically wastes redox equivalents for the enzymatic reaction and generates toxic intermediates and products for the cell, such as radical species and H 2 O 2 (Scheme 1).Consequently, the efficient transfer of FAD hq from StyB to StyA is of pivotal importance in nature as well as for biocatalytic applications of SMO.In fact, naturally fused monooxygenases acting on styrene have also been identified in bacterial strains such as Rhodococcus opacus, Nocardia farcinica and Arthrobacter aurescens. [9]owever, the specific activity of these naturally fused enzymes (initially classified as SMOs) was found to be 10 to 100 times lower than the activity of the discrete StyA and StyB from Pseudomonas sp. [10]These naturally fused enzymes are phylogenetically related to GEMs E2-type; [2b] therefore, the natural substrate should be indole instead of styrene, thus probably explaining the lower activity towards styrene.
With the aim of improving the efficiency of the epoxidation reaction catalysed by the two-component StyA-StyB system and understanding the mechanism of transfer of the FAD hq from StyB to StyA, we and other groups have created artificially fused Scheme 1.3a,5,7] SMOs using the StyA and StyB from Pseudomonas putida S12. [4,6]n particular, the artificially fused SMO from our group (Fus-SMO) showed higher storage stability than the others since the addition of FAD, glycerol or other additives was not necessary.
However, the catalytic functioning of the SMO bi-enzymatic system, and more in general of GEMs, is still matter of debate regarding the mode of transfer of the reduced flavin (FAD hq ) from StyB to StyA that can be either a diffusive and/or a channelling process.This work aims at understanding whether man-made fusions between StyB and StyA can increase the efficiency of the transfer of FAD hq from StyB to StyA by enabling improved channelling versus free diffusion.

Results and Discussion
Purification and characterisation of Fus-SMO Fus-SMO was created in our previous work by fusing the Cterminus of the N-terminal His 6 -tagged StyA to the N-terminus of StyB through a flexible linker of 30 amino acid residues.The linker consisted of a (SGGGG) 5 repetitive motif with an additional alanine residue at the front and a SGAS motif at the back (see SI, section 1 for details). [4]In the present work, highly reproducible recombinant expression and purification of His 6tagged Fus-SMO by Ni 2 + -affinity chromatography always afforded the chimeric enzyme with a FAD ox cofactor loading of 26-31 % as determined by quantitative UV-vis absorption spectroscopy (SI, Figure S1; range determined from four independently produced batches of enzyme).In this work, external FAD was not added prior cell lysis or during the dialysis of the purified enzyme.The protein yield was 40 milligrams per litre of culture, equal to 9 milligrams per gram of wet cells.The determination of the oligomeric state of Fus-SMO revealed that the enzyme is mainly present in two states in solution, as a trimer and hexamer, which are apparently in equilibrium with each other (see Figure 2d).However, as determined by SEC chromatography, the trimeric state appears to be the most abundant in solution (SI, Table S1 and Figure 2d).We also assume that the FAD ox is bound to the StyB unit because the K D of FAD ox for StyB is three orders of magnitude lower than for StyA.A model structure of the Fus-SMO was created in silico by modelling individually the components StyA, StyB and flexible linker (SI, section 5).For StyA, we used the X-ray crystal structure (PDB 3HIM) with a minor manipulation at the Nterminus (see SI, section 5.1).In contrast, the X-ray crystal structure of StyB (PDB 4F07) lacked the initial 13 amino acid residues and some flexible loops within the structure; therefore, we generated the missing portions of StyB in silico by running a homology model (see SI, section 5.2).The flexible linker was also modelled as described in SI, section 5.3.The linker shows mainly flexible loops as expected due to the high content of glycine residues, but interestingly also a small parallel β-strand generated by lateral interactions between the Thr-Ile-Ala motif at its start and the fifth serine residue within the sequence.This arrangement might allow for the required conformational flexibility, while ensuring optimal proximity between StyA and StyB in the final structure (Figure 2a-c).In fact, the components described above were fused in silico by considering the probable Fus-SMO architecture (StyA)-(StyB) 2 -(StyA) 2 -StyB that was inferred from the results of SEC chromatography (i.e. trimeric structure).Figure 2a depicts the final computational model, in which the StyA and StyB units are separated at 45-55 Å distance by the linker.The Fus-SMO model was obtained by including the structure of one StyB dimer that had two FAD molecules bound (see Figure 2b and 2c).As our experimental UV-vis spectroscopic determination revealed a cofactor loading of ca.1/3 (i.e., one molecule of FAD for each trimer of Fus-SMO; SI, Figure S1), we can infer that only one of the two sites are statistically occupied by the cofactor in the expressed enzyme.Furthermore, it is important to note that the model of Fus-SMO depicted in Figure 2a-c was only intended to provide the most likely architecture of the chimeric enzyme with a particular focus on the distance between StyB and StyA that is determined by the length and 3D-structure of the linker.
The melting point (T m ) of the Fus-SMO was also determined in Tris-HCl and in KPi buffers in presence and absence of FAD (Table 1 and SI, Figure S2).In both cases, the addition of FAD increased the T m .Although Fus-SMO is made by the fusion of two enzymes (StyA and StyB), only one melting temperature was observed, thus indicating that both enzymatic units should denature concomitantly at the same temperature.

Pre-steady-state kinetic measurements
Reductive rate of Fus-SMO with increasing concentrations of NADH (reduction of FAD ox to FAD hq ).We performed the presteady-state kinetic measurements in Tris-HCl buffer (pH 7.5, 20 mM) and at 25 °C in a stopped-flow apparatus under anoxic conditions.These pre-steady-state kinetics aimed at determining the rate for the reduction of FAD ox catalysed by the StyB unit within the engineered Fus-SMO.The kinetics were conducted using the enzyme solution obtained after purification, dialysis and concentration, thus having a FAD ox loading of ca. 30 %.However, more experiments were performed by also supplementing flavin in solution to reach a theoretical molar ratio of 1 : 1 between Fus-SMO protomer and cofactor.FAD ox bound to the Fus-SMO exhibits two maximum absorption peaks at 370 and 455 nm. [11]In these kinetic experiments, we have monitored the absorption at 455 nm within the time range from 0.001 s and 1 s.
The data reported in Table 2 and Figure 3 show that the addition of external FAD ox decreased the value of the kinetic constant for the reduction of the cofactor (for details SI, Table S2 and Figure S4).Therefore, the binding of free FAD ox to the StyB unit's active site must be the rate-determining step for the cofactor reduction.This is also corroborated by the fact that the kinetic measurements with Fus-SMO without exogenous supplementation of FAD exhibited a monophasic fit, whereas the same kinetics with the addition of FAD did not properly fit a monophasic curve and displayed an almost bi-phasic fit (see SI,  Figure.S5 and S6).This observation likely indicates that the second phase consists in the binding of free FAD to the active site and subsequent reduction by NADH.
Oxidative rate for FAD hq to FAD OOH in presence of O 2 and subsequent decomposition to FAD ox and H 2 O 2 .The rate for the oxidation of FAD hq in presence of molecular oxygen to form FAD OOH , and the rate for the further decomposition to FAD ox and H 2 O 2 were measured in the stopped-flow apparatus.While FAD ox absorbs at 370 nm and 455 nm, FAD hq does not absorb at 455 nm and only a little at 370 nm.FAD OOH absorbs significantly at 375-380 nm but not at all at 455 nm. [11]Therefore, after mixing FAD hq with dioxygen in the stopped-flow apparatus, the formation of FAD OOH was observable at 375 nm.This event must be followed by an increase of the absorbance at 455 nm due to the conversion of FAD OOH to FAD ox .In our experiments, FAD hq was prepared by reducing FAD ox (the one bound to the enzyme and, in selected experiments, also supplemented as free in solution) with 1.2 eq. of NADH.The experiments were conducted again in Tris-HCl buffer (pH 7.5, 20 mM) and at 25 °C.In 1 Tris-HCl [a] 32 36 2 KPi [a] 36 38 [a] pH 7, 50 mM.this case, the rate for the formation of FAD OOH was independent from the presence or absence of exogenously added FAD and it was in the range of 86-90 s À 1 (see Table 3).These rates are comparable to the rates reported for the natural discrete StyA-StyB system (k ox > 49 s À 1 or 104 s À 1 , [5b,6] as well as for the naturally fused StyA2B from Rhodococcus opacus 1CP (85 s À 1 ). [10]owever, the subsequent decomposition of FAD OOH was dependent from the experimental conditions.In absence of free-in-solution (added) FAD, a monophasic decomposition rate with k FAD(OOH)/FAD(ox) of 0.16 s À 1 was measured (Table 3, entry 1; Figure 4 and SI, Figure S8).In the presence of free FAD in solution to reach a 1 : 1 molar ratio between the Fus-SMO and the total FAD (enzyme-bound FAD plus free-in-solution FAD), a biphasic decomposition was observed with a slow rate of 0.17 s À 1 (comparable to the value obtained with Fus-SMO "as purified") and a faster rate of 1.8 s À 1 (Table 3, entry 2; Figure 4 and SI, Figure S8 and S9).The two different decomposition rates observed in the presence of added free FAD demonstrate that FAD exists in solution in two states with Fus-SMO.When FAD is bound to the enzyme, the enzymatic environment seems to protect the formed FAD OOH against spontaneous decomposition, whereas the free FAD hq gets reoxidised and decomposed quickly.

Oxidative rate for the conversion of FAD hq to FADH OOH in presence of O 2 and styrene subsequent decomposition
The rate for the oxidation of FAD hq to FAD OOH and the subsequent conversion to FAD OH were measured in presence of styrene as substrate by monitoring the absorption at 375 and 455 nm. [11]Styrene epoxidation can occur upon formation of the ternary complex.The kinetics were performed under the same conditions as before using Fus-SMO (50 μM) with 16 μM of FAD ox occupancy obtained after recombinant expression.FAD hq was generated in the active site via pre-incubation with NADH.Additional FAD was also supplemented in these experiments; however, analysis of these data did not result in conclusive interpretations in these latter cases (data not shown).
The ratio between active Fus-SMO (estimated to be equal to the concentration of bound FAD) and styrene was varied from 1 : 1 to ca. 1 : 30 molar ratio.At lower molar ratio between FAD hq and styrene (1 : 1 and 1 : 2), the rate for the formation of FAD OOH and further conversion to FAD OH (k FAD(hq)/FAD(OOH)/FAD(OH) = 90 s À 1 ) was essentially the same as the rate for the oxidation of FAD hq in absence of styrene, as reported in the previous section.The rate for the FAD OOH /FAD OH formation increased to ca. 120 s À 1 at increased styrene concentrations related to the FAD hq , but then decreased again to ca. 80 s À 1 when even higher concentrations of styrene were applied (see Table 4 and Figure 5, and SI section 6.3 for details).This observation can potentially signify an inhibitory effect of styrene at higher molar ratio.
The rate of the subsequent discrete dehydration step of FAD OH to FAD ox was in the range of 4-7 s À 1 , depending on the styrene concentration.Notably, this value of k dehyd .for Fus-SMO is higher than the reported values for the discrete StyA-StyB system (0.82 s À 1 ) [5b] and for the naturally fused StyA2B from Rhodococcus opacus 1CP (1.65 s À 1 ). [10]ble 3. Pre-steady-state kinetics data for the oxidation of FAD hq in presence of molecular oxygen to form FAD OOH , and further decomposition to give FAD ox and H 2 O 2 .

Determination of the coupling efficiency
Finally, we attempted to determine the coupling between the reduction of FAD ox to FAD hq catalysed by the subunit StyB of Fus-SMO at the expense of NADH and the epoxidation of styrene catalysed by the subunit StyA of Fus-SMO at the expense of O 2 (Table 5 and SI Tables S4, S5 and S6).This study could show any possible enhancement of the coupling efficiency between NADH consumption and styrene epoxidation due to the fusion of the two components of SMO and, therefore, be an indirect proof of the channeling of FAD hq from StyB to StyA during the catalytic cycle (for experimental details, see SI, section 7).The first study was performed at fixed Fus-SMO concentration without any exogenous addition of FAD ox and by varying the molar ratio between NADH and styrene in solution (measured in two independent sets of experiments at varied relative concentrations of NADH and styrene within the specified range).Within the range 100 : 25 and 25 : 100 μmolar ratios between NADH and styrene as well as at 100 : 100 μmolar ratio, the coupling efficiency was always � 100 % (Table 5, entry 1).By increasing the concentrations range up to 300 : 50 and 50 : 300 μmolar ratios and at 300 : 300 μmolar ratio, a reproducible slightly decrease of the efficiency was observed (as low as 94 % and a mean value of ca.98 % coupling efficiency; Table 5, entry 2).
In the second study, we added exogenous FAD ox to reach an equimolar amount to Fus-SMO and repeated the experiments.The addition of FAD ox led to a further decrease of the coupling efficiency for both previously defined ranges of NADH and styrene concentrations.Within the range 100 : 25 and 25 : 100 μmolar ratios between NADH and styrene as well as at 100 : 100 μmolar ratio, the coupling efficiency was in the range 89-96 % (Table 5, entry 3).By increasing the concentrations range up to 300 : 50 and 50 : 300 μmolar ratios and at 300 : 300 μmolar ratio, the coupling efficiency was again decreased further to 78-88 % in presence of the added FAD ox .;Table 5, entry 4).
All in all, these data indicate that the presence of FAD ox free in solution is detrimental for the coupling efficiency of the epoxidation reaction and results in the wastage of redox equivalents from NADH.We attribute the partial uncoupling due to the spontaneous chemical reaction between FAD hq (produced by the StyB unit of Fus-SMO) and the exogenously added FAD ox to generate two molecules of flavin semiquinone (FAD * ), which ultimately leads to the formation of H 2 O 2 and FAD ox (see Scheme 1).Therefore, it appears that the recombinantly produced form of the Fus-SMO, possessing a ca.30 % occupancy of the FAD cofactor, is optimal to ensure sufficiently high epoxidation rates, while avoiding an unnecessary overconsumption of NADH.
Moreover, we demonstrate that the fusion of the StyB and StyA components of the SMO ensures to obtain a virtually quantitative coupling efficiency in solution, a property that is not commonly attainable with the natural two-component SMO. [12]In the case of the natural SMO with the discrete components StyA and StyB, the coupling efficiency also decreases when increasing the FAD concentration.However, at  the optimal FAD concentration, the highest coupling efficiency can only be obtained at increased StyA/StyB molar ratio (> 5 : 1, mol mol À 1 ). [12]In contrast, virtually quantitative coupling efficiency is observed with Fus-SMO in which the two components StyA and SyB are in equimolar amount.These findings suggest that the fusion between StyA and StyB within Fus-SMO might enhance the channelling of FAD hq from the two subunits.We attribute this property to the proximity between StyA and StyB in solution that is determined by the length and 3D-structure of the flexible linker.Notably, the channelling (or partial channelling) of FAD hq was already postulated by Kantz et al. [12]

Conclusions
Published data shows that StyB and StyA have evolved to have a strong tendency to bind preferably FAD ox and FAD hq , respectively.These kinetic properties must also be important to enable the efficient transfer of FAD hq from StyB to StyA during the catalytic cycle.However, the mode of transfer of FAD hq is still debated as it could proceed via free diffusion and/or channelling, also depending on the conditions for catalysis.In our previous work, the fusion of StyA with StyB using a flexible linker resulted in a chimera (Fus-SMO) showing a minimised consumption of redox equivalents (NADH).This observation led us to postulate that the fusion of the two components improved the transfer of the FAD hq between StyB and StyA, so that the occurrence of the catalytically unproductive cycle to give FAD ox and H 2 O 2 was minimised.
In this work, Fus-SMO was biochemically characterised to be mainly a trimer in solution with a FAD loading of ca.1/3.Considering the most probable trimeric arrangement (StyA)-(StyB) 2 -(StyA) 2 -(StyB), we generated a model in silico.The model showed that the various components were distanced at 45-50 Å due to a loopy arrangement of the linker, which looked partly stabilised by its short parallel β-strand spatially close to the StyA component.This arrangement seems to be ideal to aid the improved transfer of FAD hq from StyB to StyA.As we deducted from the UV-vis spectroscopic analysis that one FAD molecule is bound per trimer of Fus-SMO, we assumed that only one of the two identical active sites of (StyB) 2 , as depicted in the in silico model, is statistically occupied at a given time.However, addition of exogenous FAD increased the thermostability of the enzyme, likely signifying a stabilising effect due to alternative FAD binding sites in Fus-SMO.
We characterised Fus-SMO by pre-steady-state kinetics.First, we determined the rate for the anoxic reduction of FAD ox to FAD hq at various conditions, deducing that the binding of free FAD ox must be the rate-determining step for FAD ox reduction.Once FAD ox is bound to the StyB component, it is reduced with a k red of ca.110 s À 1 , while the affinity for the binding of NADH is comparable with the natural two-component StyA-StyB.The rate for the aerobic oxidation of FAD hq to FAD OOH of Fus-SMO (ca.90 s À 1 ) was also similar to the values for the natural discrete StyA-StyB from Pseudomonas putida S12 or the naturally fused StyA2B from Rhodococcus opacus 1CP.The subsequent decom-position of FAD OOH for Fus-SMO was different in absence or presence of exogenously added FAD.We concluded that FAD OOH is protected against spontaneous elimination of H 2 O 2 when it is bound to StyA.The pre-steady-state aerobic oxidation of FAD hq to FAD OOH maintained the same rate also at low styrene concentration (up to twice molar excess of styrene to FAD).The oxidation rate increased up to 120 s À 1 at five times molar excess of styrene to FAD, but decreased again upon a further increase of the styrene concentration relative to FAD.
Finally, we determined that the coupling efficiency between NADH consumption and styrene epoxidation for Fus-SMO was worsen when free FAD ox was added in solution, signifying that exogenous FAD enables the unproductive catalytic cycle to H 2 O 2 that ultimately wastes equivalents of NADH.In contrast, the recombinantly produced Fus-SMO with its 30 % of FAD occupancy was already optimal to obtain high epoxidation rates, while avoiding an unnecessary overconsumption of NADH.In fact, we demonstrated that Fus-SMO -in which the two components StyA and SyB are in equimolar amountensures to obtain a virtually quantitative coupling efficiency in solution, a property that was not observed with the natural two-component SMOs.All in all, these findings suggest that our fusion between StyA and StyB might have enhanced the channelling of FAD hq between the two subunits.
In summary, this work elucidates some aspects regarding the transfer of the FAD cofactor between the two components of the SMO, a process that could proceed differently when the two discrete units are isolated in solution or in a cellular environment. [13]The proximity of StyA and StyB in solution via the fusion with the flexible linker might better replicate cellular conditions.Moreover, this study demonstrates how the relatively simple protein engineering technique of protein fusion can significantly improve the catalytic efficiency of a twocomponent system like SMO, even though the natural system is already applied in chemical synthesis in both laboratory and industry settings. [14]

Figure 1 .
Figure 1.The catalytic mechanism of the bi-enzymatic system (StyA-StyB) of the styrene monooxygenase (SMO): a) schematic depiction of the reduction of FAD ox catalysed by StyB, the transfer of the obtained FAD hq (i.e. FADH 2 ) to StyA, and generation of flavin hydroperoxide (FAD OOH ); b) asymmetric epoxidation reaction catalysed by StyA and regeneration of FAD ox ; (reelaborated from ref.[4] c) the flavin cycle within the catalytic mechanism of SMO.

Figure 2 .
Figure 2. a) In silico model of Fus-SMO as a (StyA)-(StyB) 2 -(StyA) 2 -StyB trimer, based on the experimentally determined oligomeric state; estimations of the spatial length of linkers, and StyA and StyB components are annotated.The model and the figures were created with Yasara (version 23.9.29; for details, see SI section 5).b) Zoom-in of the StyB dimeric unit, showing two molecules of bound FAD at two structurally identical sites; however, only one of these two sites is statistically occupied by the FAD in the recombinantly expressed enzyme as determined by UV-vis spectroscopy (Figure S1).c) Zoom-in of the FAD moiety bound between the dimeric interface of the two StyB protomers.d) Size exclusion chromatography for Fus-SMO.
K M values for NADH could not accurately be determined due to the too low required concentration of NADH.The lowest NADH concentrations were 35 and 30 μM (ca.2-time or 5-time in excess to FAD).K M values are in the range of published data for the natural StyB.

Figure 3 .
Figure 3. Kinetic plots for the reduction of FAD ox catalysed by the StyB unit within the engineered Fus-SMO.Three independent measurements were performed for every experiment using different charges of enzymes using enzymes concentrations of 50-70 μM and NADH concentrations from 30 to 200 μM.

Figure 4 .
Figure 4. Overlay of the stopped-flow traces obtained for the formation and decomposition of FAD OOH using Fus-SMO without external addition of FAD (black and grey lines for formation and decomposition, respectively) and with addition of FAD (red and purple lines for formation and decomposition, respectively).Reduced FAD [FADH 2 , obtained by the reduction with a 1.2fold excess of NADH, 32-70 μM Fus-SMO (as purified, ~30 % bound FAD) or with externally added FAD (enzyme : FAD equimolar)] was shot against buffer that contained dioxygen (buffer produced under atmospheric pressure contains around 200 μM of O 2 at room temperature).Independent duplicates have been performed.

Figure 5 .
Figure 5. Overlay of the stopped-flow traces obtained for the oxidative rates in the presence of O 2 and styrene.The red lines indicate the measurements without the addition of styrene at 375 nm and 455 nm, respectively.The black/grey lines indicate the measurements in the presence of styrene to obtain a molar ratio of styrene:Fus-SMO of 1, 15 and 28.Independent duplicates have been performed with reduced FAD [FADH 2 , obtained by the reduction with a 1.2-fold excess of NADH, 30-70 μM Fus-SMO (as purified, ~30 % bound FAD) or with externally added FAD (enzyme:FAD equimolar)].That was shot against buffer that contained dioxygen (buffer produced under atmospheric pressure contains around 200 μM of O 2 at room temperature) and different concentrations of styrene (from 0 to 500 μM).

Table 2 .
Pre-steady-state kinetics data for the reduction of FAD ox catalysed by the StyB unit within the engineered Fus-SMO.

Table 4 .
Pre-steady-state kinetics data for the oxidation of FAD hq in presence of molecular oxygen and styrene to form FAD OOH , and further decomposition to give FAD ox and H 2 O without the addition of free FAD.

Table 5 .
Determination of the coupling efficiency between FAD ox reduction and styrene epoxidation catalysed by the components StyB and StyA, respectively, within Fus-SMO.