Robert
Röllig
ab,
Caroline E.
Paul
c,
Magalie
Claeys-Bruno
d,
Katia
Duquesne
a,
Selin
Kara
b and
Véronique
Alphand
*a
aAix Marseille Univ, CNRS, Centrale Marseille, iSm2 UMR 7313, Marseille, France. E-mail: v.alphand@univ-amu.fr
bAarhus University, Denmark
cDelft University of Technology, The Netherlands
dAix Marseille Univ, Univ Avignon, CNRS, IRD, IMBE UMR7263, Marseille, France
First published on 22nd March 2021
Two-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer–Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer–Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case.
Oxidative enzymes are capable of catalysing a large range of reactions, but often require cofactors, which have to be recycled to enable their catalytic usage. Among these enzymes, the class of oxygenases is immensely diverse5–8 and contains inter alia flavoprotein monooxygenases (FPMOs), a family in which are found enzymes that catalyse BV oxidation. These monooxygenases belong to the groups A, B or C of FPMOs9 and are single- or two-component enzymes (Scheme 1), respectively called type I and type II Baeyer–Villiger monooxygenases (BVMOs).9–11
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Scheme 1 Flavin-dependent single- and two-component monooxygenase systems. (a) One-component FPMO system: the reductase and oxygenase domains are gathered in a single protein. Flavin is tightly bound as a prosthetic group in the active site of the enzyme. (b) Two-component FPMO system: reductase and oxygenase components are in two separated proteins. The flavin reduction catalysed by the reductase is driven by NAD(P)H oxidation, whereas flavin-H2 oxidation is driven by the oxygenase. The mode of flavin-H2 transport depends on the two-component monooxygenase, achieved either by free diffusion or via reductase-oxygenase mediation.12 |
Despite their structural differences, the one- and two-component oxygenase systems share a dependency on the expensive nicotinamide adenine dinucleotide cofactor (NAD(P)H). Moreover, the stability of NAD(P)H can become a hurdle (under basic conditions), as the adenine dinucleotide moiety is susceptible to hydrolysis.13–15
Synthetic nicotinamide analogues (nicotinamide coenzyme biomimetics, NCBs) address both obstacles, as they can be synthesized at lower costs while remaining stable.16 Moreover, they have been established to replace their natural templates for numerous flavin-dependent enzymes17–19 with a flavin shuttled electron mediation.20–22
Most of the studies on enzymatic BV oxidation focused on one-component FPMOs from group B (also known as type I BVMOs). These enzymes are available in high numbers in protein databanks, easily detectable by consensus sequence research23 and subsequently more frequently investigated and applied in many catalytic processes.3,4,24–26 In contrast, two-component systems referred to as type II BVMOs (from group C of FPMOs) are rare. Only two representatives are known so far, 2,5-diketocamphane 1,2-monooxygenase I (2,5-DKCMO; EC 1.14.14.108) and 3,6-diketocamphane 1,6-monooyxgenase (2,6-DKCMO; EC 1.14.14.155),27–32 probably because a two-component system is not a trivial biocatalytic arrangement to identify. The mechanism of hydride transfer and the balance of the reductase and oxygenase reactions are still unknown. Consequently, the question of whether the oxygenase can work independently of the reductase component and nicotinamide cofactor remains to be determined.
In this in vitro study, we showed the promising catalytic capacity of the oxygenase component and established its mode of reaction. We report an innovative and simplified reductase-free enzymatic system for BV oxidation, driven by NCB hydride donation for flavin mononucleotide (FMN) reduction and further electron mediation through the flavin. The purified type II BVMO 2,5-DKCMO from Pseudomonas putida ATCC 1745330,31,33–35 was applied in a reductase-free fashion and compared with a reductase-oxygenase two-component system. The regio- and enantioselective BV oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one (rac-1) was used as the model reaction. The reductase-free system was further investigated focusing on potential limits of the approach. Design of Experiments (DoE) was executed to estimate the impact of the seven reaction parameters and optimize the final system. The utilization of NCBs allowed us to address the previous question and demonstrated the compatibility of the monooxygenase with artificial hydride donors without a loss of activity. Finally, the tunability of the system to avoid flavin degradation is discussed and the concept of flavin recycling as the adjusting wheel of two-component systems is highlighted and validated by carrying out a complete reaction.
The type II BVMO applied in this study is the well-known 2,5-DKCMO from P. putida ATCC 17453 (NCIMB 10007),30,31,33–35 which was produced via heterologous expression as an E. coli-codon-optimized version with an N-terminal His-tag for affinity chromatography purification (see ESI section A for further details†). A fast substrate profiling showed that rac-bicyclo[3.2.0]hept-2-en-6-one 1, an established substrate for the evaluation of enantioselective BV oxidation37 (Fig. 1), displayed the highest conversion rate among the assayed compounds (see ESI, Fig. S4†). The biotransformation of rac-1 by 2,5-DKCMO was already investigated by Lau and co-workers,34 as well as the group of Bornscheuer,35,38,39 but no complete characterization of the reaction had been made. Thus, we selected this compound as the model substrate for our study.
The two partially water-soluble and stable NCBs, 1-benzyl-1,4-dihydronicotinamide (BNAH), and 1-(2-carbamoylmethyl)-1,4-dihydronicotinamide (AmNAH, caricotamide), were selected. To ensure a homogeneous reaction system, both NCBs were supplied from a stock solution in DMSO. The first experiments, with both NCBs and without reductase, showed a slow enantioselective conversion of ketone 1 with an increase of the enantiomeric excess in favour of the (+)-enantiomer and a preferential formation of (+)-(1R,5S)-2-oxabicyclo[3.3.0]oct-6-en-3-one 2, also called (+)-normal lactone. The reaction system is shown in Scheme 2. Both NCBs provided the hydride donation for the reduction of the flavin cofactor FMN before FMNH2 diffusion into the active site of the oxygenase. However, a difference of performance was observed as shown in Fig. 2 and AmNAH appears as the most suitable hydride donor. Both a higher ee of the substrate and a higher conversion were observed at the two tested concentrations (10 and 50 mM).
The preliminary results did not show the full potential of the approach. The presence of reduced and oxidized flavin, as well as oxygen in the aqueous medium, is a potentially delicate composition.42–46 In absence of the catalase, a change of the colour of the reaction from pale yellow to orange was observed with AmNAH as shown in Fig. 3. This indicates the formation of anionic semiquinone flavin radicals and other flavin decomposition products, due to an uncoupling of the reduced flavins into radicals and hydrogen peroxide.47,48 H2O2 is known to be formed with flavoprotein monooxygenases, including two component styrene monooxygenase and halogenases.20,21 On the other hand, it is also known to oxidize substrates as ketone 1 in racemic lactone 2.49 In our case, the addition of catalase improved the biotransformation (Fig. 3) as illustrated by the complete disappearance of the (−)-ketone 1 in six hours along with a better ee of normal lactone 2 (82% and 55% respectively with and without catalase after two hours of reaction). Consequently, it can be concluded that catalase addition alleviates the unselective chemical BV oxidation by H2O2. In this regard, the stability of the enzyme in the presence of H2O2 was also evaluated.‡ After its storage in up to 50 mM H2O2 for four hours, no significant decline of activity was observed (data not shown), confirming the great stability of the oxygenase.
To determine the behaviour of our model type II BVMO system in the presence and absence of a reductase, purified flavin reductase (Fre) from E. coli was selected to act as the reductase counterpart. This enzyme was previously described as a suitable supplier of reduced FMN for 2,5-DKCMO.30 The Fre enzyme was applied in our experiments in an equimolar amount (compared to the monooxygenase) using NADH as the hydride donor. To evaluate the potential impact of Fre on the catalytic performance of the reaction system, identical setups without Fre served as control experiments (Fig. 4).
Surprisingly, in terms of substrate conversion, the reductase-free system applying AmNAH as the hydride donor outperformed the reductase setup using NADH over time (see Fig. 4a). Similar results were obtained when five times more Fre enzyme was applied§ (data not shown). The NADH-driven Fre-system did not exceed a final conversion of 63 ± 0% (reached in less than two hours) in the equimolar Fre system, or 56 ± 2% (reached after three hours) when Fre was applied in five times excess, respectively. The reaction system without the reductase, driven by the direct FMN reduction via AmNAH, reached nearly full conversion (97 ± 1%) after six hours. The evolution of ee of ketone 1 was also in agreement with this observation.
Additionally, we compared the two systems with the reaction only driven by NADH (without Fre). To succeed in comparing the enantioselectivity of the three complex systems, the effect of different FMN reduction rates has to be eliminated. Thus, Fig. 4b shows the substrate ee as a function of the conversion. The plotted curves of the reductase-free reaction setup with AmNAH, as well as with NADH were perfectly supposable, and in agreement with the experiment associating the reductase and NADH.
With these experiments, we demonstrated the independence of 2,5-DKCMO from any flavin reductase enzyme exemplary by its independence from Fre. The oxygenating component of the chosen type II BVMO system maintained its enzymatic capacity in the absence of the reductase. Moreover, the nicotinamide cofactor was substituted by AmNAH, demonstrating that the hydride donor for in situ FMN reduction can be easily replaced. Therefore, we conclude that the mechanism of reduced flavin transfer in this two-component monooxygenase system does not require a protein–protein interaction but is achieved passively by free diffusion.
The impact of different reaction parameters in the reductase-free setup was tested using a DoE (Fig. 5) comprising NCB type and concentration, FMN and oxygenase concentration as well as temperature and buffer type, and pH (for DOE details, see ESI, Table S1†).
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Fig. 5 Screening by DoE of parameter effects on reductase-free 2,5-DKCMO-mediated enantioselective BV oxidations of rac-1. The (i) enantiomeric excess of the ketone 1 at three hours and (ii) total conversion after 22 hours were selected to calculate the DoE output. The parameters are shown to the left, the levels are shown to the right: e.g. [FMN] is the parameter, and 25–150 μM are the levels. The length of the bars represents the impact of the parameter levels on ketone ee and conversion. Greater effects are shown by larger bars. Blue numbers show some changes in the values of outputs compared to the reference level (black bars and dashed lines). Further explanations are provided in the ESI.† Experimental conditions: catalase was added in great excess (2.5 mg mL−1), NCBs were supplied from a DMSO stock solution (1 M) to a total volume of 0.5 mL, other parameters were modified as shown in the figure. |
The ee of the ketone after three hours and the final conversion were chosen as data for the DoE calculation. They allowed respectively an insight into (i) the reaction rate (activity) of the system in the early stages of the reaction, by monitoring the enantioselectivity of the enzyme, and (ii) the durability of the system by monitoring the capacity of the enzyme to transform the substrate in 22 hours. The latter one is of particular interest because it depends on the process stability of all species present in its environment.
The DoE results are shown in Fig. 5. It is important to note that the length of bars in the figure is not proportional to the quantitative outcomes, but represents the influence of a given parameter on the outcomes. From the DoE results (Fig. 5), we concluded that the buffer (both nature and pH) had a relatively small effect on both outputs, and hence on the whole system. Unsurprisingly, the temperature and oxygenase concentration affected the system during the entire reaction. All other tested parameters affected the response largely but with varying degrees of intensity in the early and later states. The same observation was observed for the FMN concentration, but with a greater effect of lower concentrations at the early stage to a lower impact in the late stage.
The NCBs revealed a clear influence of their nature and concentration on the performance of the system. Interestingly, the impact of the N-substituent type, an amide (AmNAH) or phenyl group (BNAH), was the strongest parameter at the early stage of the reaction (ketone ee at three hours), as shown by the AmNAH bar being considerably longer than its BNAH counterpart. We attribute the early stage-impact to the dissimilar flavin reduction rate (NCB-driven hydride transfer) evoked by different (electro)chemical characteristics e.g. redox potential of the NCBs,57 but also their chemical stability in aqueous solution. Although BNAH is the stronger reducing agent, its half-life time in aqueous media (approx. 1.5 hours) is more than ten times shorter compared to AmNAH.16,41 At the end of the reaction, the impact of both NCBs decreased, as the rate of hydride transfer to FMN became negligible, and therefore insignificant. Hydride donation was not only affected by the type of hydride donor (NCBs) but also by the concentration, as we observed an impact of this parameter at the early stage and the end of the reaction. Interestingly, this shifts over time with an increasing impact of lower concentrations at the later stage of the reaction. Thus, lower NCB concentrations did not result in higher conversion. However, it must be noted that the interpretations gained from the DoE are only valid within its boundaries, e.g. an impact of a pH value below 7.5, or above 8.5 might be considerable.
The analysis of DoE results shows that all parameters with a great impact on the reaction system are associated directly with the FMN/FMNH2 cycle (see Fig. 6). This applies to the reduction of the flavin, e.g. the type and concentration of the NCBs (see Fig. 6 in blue), but also to its enzymatic FMNH2 (re)oxidation for which the oxygenase concentration (see Fig. 6 in red) is crucial.
The temperature was also identified as a relevant parameter in the system, which is in accordance with our interpretation, as it affects both flavin reduction and (re)oxidation, albeit to a different extent (reduction = chemically versus oxidation = enzymatically). In some experiments, an increased chemical oxidation of ketone but no bioconversion was observed. Therefore, the impact of the parameter on both the chemical and chemoenzymatic performance of the reaction system was also evaluated.
As a consequence of the identification of the key role of the flavin and its function as the adjusting wheel of the reaction, we assessed the FMN reduction/(re)oxidation cycle to be crucial for a (long-time) catalysis. The reduction and oxidation rates of the flavin need to be balanced to ensure the greater stability of the system's components, in particular, to minimize the instability of the reduced flavin in the presence of oxygen.
Among the tested substrates, 2,5-DKCMO showed the highest activity towards the model substrate rac-1 with a maximum conversion of approx. 75% (Fig. 7a), followed by rac-4 with >35% (Fig. 7b) with an exclusive conversion of (1R,4R)-(+)-camphor. rac-5 is a rather poor substrate achieving conversions less than 20% (Fig. 7c) within the first two hours under the given conditions. For all tested substrates we observed greater conversions (Fig. 5a–c) at higher temperatures. For rac-1, low and medium FMN concentration (up to 20 μM) resulted in better performances. Similar results were observed with rac-4 (or more precisely (1R,4R)-(+)-camphor) and rac-5 despite significantly lower rates.
Regarding the ee of the substrates (Fig. 7d–f) we observed (i) an effect of the temperature on the rac-1, resulting in highest ee values at low to moderate temperatures and (ii) an effect of the FMN concentration on rac-4 with a clear trend for higher ee at low FMN concentrations. In this context, we expect the optimal FMN concentration for the reaction to be lower than our tested range. The results of rac-5 however, are difficult to evaluate due to the low conversion in the DOE condition window.
Summarizing the results of the second DoE, we observed the general trend for better enzymatic performance at lower to mediate temperatures and flavin concentrations.
These data support our hypothesis of the flavin/flavin-H2 cycle being the bottleneck and hence the major adjusting wheel in the reductase-free setup. It is also noticeable that the optima for the three compounds are slightly different, which reflects the influence of different enzymatic oxidation rates. Therefore, in a virtually optimized reaction setup, we expect to accomplish experimental conditions, in which flavin reduction and the (enzymatic) (re)oxidation are appropriately balanced.
The BV oxidation of each enantiomer of 1 offers theoretically two possibilities, as shown in Fig. 1. (−)-Ketone 1 can be transformed into either the so-called (+)-normal lactone 2 or (−)-abnormal lactone 3, while (+)-ketone delivers the opposite enantiomers. The temperature of the biotransformation was lowered to 14 °C for a better vision of the evolution of each enantiomer. Indeed, as suggested in Fig. 7d and in accordance with a behaviour already described for various enzymes,58,59 enantioselectivity was favourably affected by a drop in temperature. Under our reaction conditions, 2,5-DKCMO converted (−)-(1S,5R)-enantiomer 1 was first obtained as shown in Fig. 8. The beginning of the bioconversion was therefore characterized by the formation of (almost exclusively) (+)-normal lactone 2. This demonstrates the high enantio- and regioselectivity of the reaction. Surprisingly, once the (−)-(1S,5R)-1 was (almost) consumed, the oxygenase converted also the (+)-(1R,5S)–1 at similar rates compared to the enantiomeric counterpart. This conversion gave birth to both (−)-normal lactone 2, as well as the (+)-abnormal lactone 3. To the best of our knowledge, such sequential oxidation of enantiomers of the rac-1 has only been described for the one-component phenylacetone monooxygenase (PAMO) with non-natural FAD derivates.60
GC analyses of the three ketones and their corresponding lactones were carried out on a Cyclosil-B column (Agilent J&W GC columns). Product identification was ensured by the comparison of GC retention times with those of authentic samples (see ESI A†).
The reductase-free setup of the redox system finally demonstrated that the transport mechanism of reduced flavin to the oxygenase in this study proceeded by free diffusion. This reductase-independent enzymatic BV oxidation enables the reduction of the complexity of this two-component enzyme. In addition, it questions the terminology of the system(s), since the reductase might only be required in the natural (in vivo) context, but can be removed in artificial systems, still maintaining the oxygenase functionality. 2,5-DKCMO might be characterized as one component with the ability to utilize (hydroquinone) electron mediators, or flavin hydroquinone-dependent monooxygenase. Therefore, for other representatives of two-component FPMOs, each oxygenase component could advantageously be investigated separately from its reductase counterpart.
We gave herein an additional example of the synthetic potential of two-component flavoprotein monooxygenases. The proof of the independence of 2,5-DCKMO from any reductase enzyme is the foundation of further reductase-free bioconversion concepts for BV oxidations. Although the enzyme in this study has a narrow substrate scope, we are convinced our work will encourage the search for more promiscuous enzymes of this family.
We see the great potential of similar systems, applying alternative FMN reduction strategies directly mediated in the solution, to elaborate simplified and efficient enzymatic redox reaction systems. Such a reductase-free system, associated with potentially cost-efficient hydride donors can serve as a promising platform. Thus, innovative and sustainable flavin-dependent biotransformations driven by in situ flavin reduction, as for example with transition metal-catalysed hydride transfers, can be target applications for further studies.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ob00015b |
‡ Assay conditions: 50 μL mL−1 oxygenase, 50 μM FMN, 25 mM NADH as the hydride source, 50 mM Tris-HCl buffer at pH 7.5, 20 °C and 160 rpm. |
§ Five times excess compared with the monooxygenase using the same NADH concentration. |
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