A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6–9) and temperatures (40–60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol−1 K−1. The KM values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the kcat values were 206.79 s−1 and 202.93 s−1, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min−1 mg1, the highest activity value described for a CPR up to date (3.2–4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst.


Introduction
Nowadays, an alarming increase of seawater pollution (aromatic hydrocarbons, nitrogenous compounds, and heavy metals, among others) is observed. Because chemical degradation of these compounds may produce negative effects on marine living organisms [1], the use of whole cells, enzymes or immobilized biocatalysts to eliminate these toxic compounds (so called bioremediation) is gaining ground. Bioremediation is an effective and eco-friendly solution for these environmental problems [2][3][4][5].
To fulfil the safety requirements of current environmental legislation, the development of new environmentally friendly and cost-effective strategies is needed [6,7].
In this sense, the enzymatic degradation of marine environmental pollutants has come up as a promising alternative that offers high catalytic efficiency and specificity [8][9][10][11].
Micro-organisms and enzymes from contaminated marine environments may be of particular interest, as they can function in a wide range of pH and temperature values, in the presence of metal ions, and even at high concentrations of organic solvents [12][13][14].
Cytochrome P450 enzymes (also named CYP450 or CYP) are a large family of hemoproteins that perform different type of oxidation reactions in presence of molecular oxy- Remarkably, LkCPR exhibited a specific activity much higher than the CPRs from other marine organisms; e.g. 8 times higher than ScCPR (60 IU/mg) [26], 9.1 times higher than LsCPR (52.6 IU/mg) [27], 12.6 times higher than CPR from Rainbow trout (RtCPR, 38 IU/mg) [28] and 20.6 times higher than CPR from Sparus aurata (SaCPR, 23.3 IU/mg) [28]. More interestingly, to our understanding, LkCPR is the most active reported CPR, showing an enzymatic activity that is 3.2-and 4.7-fold higher than the CPRs from Porcine Polymorphonuclear Leukocytes (152 IU/mg) [29], Saccharomyces cerevisiae (150 IU/mg) [30] and Rattus norvegicus (102 IU/mg) [31], the ones previously described as the most active CPRs. We are analyzing the structural causes for this interesting result (work in progress).
SDS-PAGE gel of the purified enzyme showed a single band around 75 kDa ( Figure 1). This molecular weight is similar to that of CPRs from the fishes Liza saliens (LsCPR, Mw 77 kDa [27]) and Alburnus tarichi (Lake Van fish, AtCPR, Mw 70 kDa [32], but is lower than the one of the CPR monomer from the marine fish Stenotomus chrysops (ScCPR) (Mw 82.6 kDa) [26].
SDS-PAGE gel of the purified enzyme showed a single band around 75 kDa ( Figure  1). This molecular weight is similar to that of CPRs from the fishes Liza saliens (LsCPR, Mw 77 kDa [27]) and Alburnus tarichi (Lake Van fish, AtCPR, Mw 70 kDa [32], but is lower than the one of the CPR monomer from the marine fish Stenotomus chrysops (ScCPR) (Mw 82.6 kDa) [26].
(a) (b) Figure 1. Liza klunzingeri fish samples collected from the northern coasts of the Persian Gulf, in Bandar Abbas and after purification of the novel NADPH-dependent cytochrome P450 reductase (for more details see The native molecular weight of LkCPR was found to be approximately 75 kDa, determined by Sephacryl S-200 gel filtration chromatography using an elution calibration curve of standard proteins (Figure 2A). This molecular weight was estimated from comparison of the electrophoretic mobility of LkCPR with the mobilities of marker proteins ( Figure  2B). These results reveal that LkCPR is a monomer in solution, similar to yeast cytochrome C reductase (a monomer of 70.0 kDa) [30], the only reported monomeric CPR. However, both CPRs display a different oligomeric state than other reported CPRs (e.g. dimeric CPR from Trypanosoma cruzi) [33]. The native molecular weight of LkCPR was found to be approximately 75 kDa, determined by Sephacryl S-200 gel filtration chromatography using an elution calibration curve of standard proteins (Figure 2A). This molecular weight was estimated from comparison of the electrophoretic mobility of LkCPR with the mobilities of marker proteins ( Figure 2B). These results reveal that LkCPR is a monomer in solution, similar to yeast cytochrome C reductase (a monomer of 70.0 kDa) [30], the only reported monomeric CPR. However, both CPRs display a different oligomeric state than other reported CPRs (e.g. dimeric CPR from Trypanosoma cruzi) [33].

Effect of pH on LkCPR Activity and Stability
The enzymatic activity of NADPH-dependent cytochrome P450 reductase remained almost unaltered in a broad pH range (from 5 to 10 the relative activity was >70%), with a maximum value at pH 8 ( Figure 3A). These results are similar to results published using other marine fish CPRs, such as LsCPR (90% relative activity in the pH range 7-8) [27] or AtCPR (82% relative activity in the pH range 7-8) [32], or the CPR from green microalga Botryococcus braunii (>90% relative activity in the pH range 7-8) [34]. In addition, LkCPR also displayed around 40% of relative activity at pH 2 and pH 12, values much higher than those reported for other CPR enzymes at these pH values. This activity at extreme pH values may have interest in bioremediation of contaminated sea areas, where the pH values may become extreme under such harsh conditions. Stability was also studied at these extreme pH values. Figure 3B shows a drastic loss of enzyme activity when the enzyme was incubated 15 min at pH 3 (<50% retained activity). Higher stability was found when the enzyme was incubated at pH 12 (60% retained activity after 30 min).

Effect of pH on LkCPR Activity and Stability
The enzymatic activity of NADPH-dependent cytochrome P450 reductase remained almost unaltered in a broad pH range (from 5 to 10 the relative activity was >70%), with a maximum value at pH 8 ( Figure 3A). These results are similar to results published using other marine fish CPRs, such as LsCPR (90% relative activity in the pH range 7-8) [27] or AtCPR (82% relative activity in the pH range 7-8) [32], or the CPR from green microalga Botryococcus braunii (>90% relative activity in the pH range 7-8) [34]. In addition, LkCPR Stability was also studied at these extreme pH values. Figure 3B shows a drastic loss of enzyme activity when the enzyme was incubated 15 min at pH 3 (<50% retained activity). Higher stability was found when the enzyme was incubated at pH 12 (60% retained activity after 30 min).

Effect of Temperature on LkCPR Activity and Stability
As shown in Figure 4A, LkCPR exhibited high activity (<70% relative activity) in a temperature range from 40 • C to 70 • C, with a maximum value at 50 • C. Moreover, the enzyme was also quite active even at 90 • C (>30% relative activity). LkCPR showed an optimum temperature similar to that of AtCPR [32], and higher than the values reported for CPR from the green microalga Botryococcus braunii (41 • C) [34]. Figure 4B shows that LkCPR showed an unusually high thermal stability even at 80 °C, conditions where the enzyme retained about 60% of the initial activity after 30 min. Furthermore, LkCPR kept 70% of the initial activity after 10 min of incubation at 90 °C. That way, LkCPR exhibits a much higher thermal stability than other purified NADPHdependent cytochrome P450 reductases, such as those from Bacillus megaterium (only 50% activity was retained after 10 min at 54 °C) [35], Capsicum annuum (totally inactivated after 10 min at 70 °C) [36], Rattus norvegicus (totally inactivated after 2 min at 60 °C) [37]. In fact, we have not found reports on CPR with a higher stability than this new LkCPR.

Steady-State Kinetics
The calculated KM values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, respectively ( Table 2). The KM values for cytochrome C exhibited by LkCPR are lower than those reported for other marine fish CPRs, such as AtCPR (12.82 μM for cytochrome C, and 5.20 μM for NADPH) [32] or ScCPR (24 μM for cytochrome C, and 14 μM for NADPH)  Figure 4B shows that LkCPR showed an unusually high thermal stability even at 80 • C, conditions where the enzyme retained about 60% of the initial activity after 30 min. Furthermore, LkCPR kept 70% of the initial activity after 10 min of incubation at 90 • C. That way, LkCPR exhibits a much higher thermal stability than other purified NADPHdependent cytochrome P450 reductases, such as those from Bacillus megaterium (only 50% Mar. Drugs 2023, 21, 99 7 of 12 activity was retained after 10 min at 54 • C) [35], Capsicum annuum (totally inactivated after 10 min at 70 • C) [36], Rattus norvegicus (totally inactivated after 2 min at 60 • C) [37]. In fact, we have not found reports on CPR with a higher stability than this new LkCPR.

Steady-State Kinetics
The calculated K M values for cytochrome C and NADPH were 8.83 µM and 7.26 µM, respectively ( Table 2). The K M values for cytochrome C exhibited by LkCPR are lower than those reported for other marine fish CPRs, such as AtCPR (12.82 µM for cytochrome C, and 5.20 µM for NADPH) [32] or ScCPR (24 µM for cytochrome C, and 14 µM for NADPH) [26], or the algae BbCPR (11.7 µM for cytochrome C, and 9.4 µM for NADPH) [34]. k cat values were 202.93 s −1 and 206.79 s −1 calculated using for cytochrome C and NADPH respectively (calculated V max values were 25.96 µM min −1 (changing cytochrome C concentration) and 26.73 µM min −1 (changing [NADPH] concentration)). This coincidence of V max using saturating concentrations of each of the substrates confirms the reliability of the results. According to the Arrhenius plot in the range 20-50 • C (data not shown), the activation energy (E a ) of the enzyme in the reaction was determined to be 16.33 kcal mol −1 K −1 . The influence of temperature on the reaction rate does not provide too much information about the reaction mechanism of the enzyme. However, they can indicate alterations of the enzyme conformation and catalytic performance [7]. In Table 3, the values of the activation enthalpy (∆H # ), the activation free energy (∆G # ), and the activation entropy (∆S # ) are also shown. The low enthalpy value of cytochrome P450 reductase from Liza klunzingeri suggests a very efficient production of the transition state. Moreover, the low ∆G # value points to a great tendency of this transition state to give the corresponding products. The change in Gibbs free energy (∆G # ) is a good indicator of the feasibility of chemical reaction, i.e., the transformation of ES complex into the corresponding products [7]. The great affinity of the enzyme towards its substrates was confirmed by the activation free energy of substrate binding (∆G # E-S ) and the free energy of the formation of the activation complex (∆G # E-T ) (1.36 and −2 kcal mol −1 , respectively).

Materials
All reagents were purchased from Merck (Darmstadt, Germany).

Collecting Samples and Extraction of Protein Extracts from Liver Microsomal Cells
The specimens used in this study (15 samples of Liza klunzingeri fish, with an approximate weight of 300 g per sample) were captured in September 2017 from the northern coasts of the Persian Gulf, in Bandar Abbas. The collected fish were washed with marine water and packed in clean plastic bags and conserved in ice, transported to the laboratory, and frozen at −80 • C. They were washed with distilled water and the liver samples were extracted by surgery. The liver samples were washed with 20 mM KCl/2 mM EDTA (pH 7.5). All subsequent steps were carried out at temperatures between 0 • C and 4 • C.
The liver samples were homogenized in 50 mM sodium phosphate buffer, pH 7.5, containing 0.5 mM ε-ACA, 0.5 mM PMSF and 5 mM EDTA. This solution was centrifuged at 20,000 rpm for 30 min at 4 • C and the supernatant was collected. This was centrifuged again at 25,000 rpm for 60 min at 4 • C. The final precipitated microsomes were resuspended in a minimum amount of 20 mM KCl/2 mM EDTA (pH 7.5). After a second centrifugation the microsomal precipitate was resuspended in a minimum aqueous solution of 10% (v/v) glycerol/2 mM EDTA. This suspension was diluted in 50 mM sodium phosphate (pH 7.5), containing 20 µM BHT, 0.5 mM PMSF, 5 mM EDTA, 0.1 mM DTT, 0.5 mM ε-ACA, 0.5% (w/v) Na-cholate, 0.5% (w/v) Emulgen 913 and 25% (v/v) glycerol. The suspension containing microsomes was incubated in Emulgen 911 and centrifuged at 4 • C for 60 min at 25,000 rpm. The yellow supernatant was separated from the sediment and kept at 4 • C.

Ammonium Sulfate Precipitation
The yellow solution obtained above was poured into a small beaker, and the beaker was placed in an ice container and submitted to magnetic stirring. Then, solid ammonium sulfate was slowly added at 4 • C up to 20% saturation. The suspension was centrifuged at 12,000 rpm for 20 min and solid ammonium sulfate was added to the above supernatant to reach 85% ammonium sulfate saturation and stirred at 4 • C. After a new centrifugation, the precipitate was resuspended in 50 mM Tris-HCl buffer at pH 7.5 and dialyzed against the same buffer for 24 h at 4 • C, performing three dialysis buffer changes (one every 8 h).

Purification and Determination of Molecular Weight
The dialyzed protein samples were subjected to ion exchange chromatography using a DEAE−Sepharose fast flow column (Sigma-Aldrich, St. Louis, MO, United States). The column (2.5 × 6.5 cm) was pre-equilibrated with 50 mM sodium phosphate buffer at pH 7.5 (buffer A) and then the sample was applied at a flow rate of 0.5 mL/min. Then, the column with the ionically exchanged proteins was extensively washed with buffer A until no protein was detected in the outlet. With a linear gradient of 0-1 M NaCl added to the buffer A, using 0.5 mL/min flow rate the bound proteins were eluted. The protein elution was followed by UV-vis spectroscopy at 280 nm. The activity of the different samples was assayed using cytochrome C as substrate (standard assay).
The fractions exhibiting activity were collected, dialyzed against buffer A, concentrated by an Amicon filter (Millipore Cork, Biochrom GmbH, Danvers, MA, United States), and subjected to re-chromatography in the same column as described above. The fractions exhibiting enzymatic activity were selected and dialyzed versus buffer A, concentrated, and applied to a 2 ,5 -ADP Sepharose 4B column, pre-equilibrated with buffer A. The bound proteins were eluted using an NaCl gradient of 0-1 M in buffer A, using a flow rate of 0.5 mL/min. After the elution, fractions with enzymatic activity were collected, dialyzed and concentrated as described above. Finally, purified protein was dialyzed against 50 mM Tris-HCl buffer at pH 7.5 for 24 h and stored at −20 • C prior to use.
To determine the oligomer state of enzyme in solution, gel filtration chromatography was performed. The purified enzyme was applied to a Sephacryl S-200 column, preequilibrated with 50 mM potassium phosphate buffer (pH 7.5), at 0.5 mL/min flow rate. The column was calibrated using the following four standard proteins: alcohol dehydrogenase (150 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (30 kDa), and cytochrome C (12.4 kDa). The plot Ve/Vo versus log molecular weight for these proteins was used to determine the molecular weight; in this plot, Ve and Vo are the volume of elution for each protein and the volume of void determined by blue dextran, respectively. SDS-PAGE was performed using a 15% polyacrylamide slab gel equilibrated with 25 mM Tris-HCl buffer at pH 8.6 containing 0.1 (w/v) % SDS. Protein concentration was determined following Bradford's method using bovine serum albumin (BSA) as standard [38].

Enzymatic Activity Assay
The activity of LkCPR was determined by monitoring the increase of absorbance at 550 nm, which is related to cytochrome C reduction [39]. In this assay, NADPH-dependent cytochrome P450 reductase (CPR) takes electrons from NADPH and transfers them to cytochrome C. The reaction was performed using 0.36 mg of pure protein solution in 1 mL of 0.3 M potassium phosphate buffer (pH 7.5) containing 50 mM of cytochrome C at room temperature [34]. The extinction coefficient of ferrous to ferric cytochrome is described to be 19.6 mM −1 cm −1 [40,41]. One activity unit (IU) was defined as the amount of enzyme (in mg) which catalyzes the reduction of 1 µmol of cytochrome C per min under the above indicated assay conditions.

Effect of Temperature on Enzyme Activity and Stability
The influence of temperature on the enzymatic activity was determined using the standard assay in the 10-90 • C temperature range, at pH 7.5. Moreover, the thermal stability of LkCPR was determined by incubating the purified enzyme in 50 mM phosphate buffer, pH 7.5, at 80 • C and 90 • C for a period of 60 min. During incubation, samples were taken at different time periods, and after cooling, the activity was measured using the standard assay.

Effect of pH on Enzyme Activity and Stability
The influence of pH on LkCPR activity was analyzed employing the standard assay determining the enzyme activity from pH 2 to 12, mixing equal amounts of 50 mM sodium acetate, 50 mM sodium phosphate, 50 mM Tris-HCl and 50 mM sodium carbonate to counteract the buffer nature effect on enzyme activity. In stability studies, LkCPR was incubated at room temperature at pH 3 and pH 12 for 60 min, and the activity was measured using the standard assay.

Determination of Kinetic Parameters
The K M , k cat , and V max were calculated under standard conditions by determining the enzyme activity at different concentrations of one of the substrates (10-100 µM cytochrome C or NADPH), while using saturation concentration for the other substrate (50 µM). Apparent K M , k cat , and V max were calculated assuming Michaelis-Menten kinetics using the GraphPad Prism 5 software [42].

Calculation of Thermodynamic Parameters
The constants of inactivation (k inact ) and enzyme activity (k cat ) were utilized to calculate the activation energy following Arrhenius equation [43].
where A is a pre-exponential factor related to the molecular collision frequency and steric effects, k (s −1 ) is the rate constant at a specific temperature T (K), E a is the activation energy of the reaction and R is the gas constant (8.314 J mol −1 K −1 ). Representing ln k as a function of 1/T produces a straight line with −E a /R as slope. The thermodynamic parameters of activation were calculated as previously stated [44]: where k cat (s −1 ) is the rate constant at T (K), h is Planck's constant (6.6256×10 −34 J s) and k B is the Boltzmann constant (1.3805×10 −23 J K −1 ).

Conclusions
Herein we report for the first time, the purification and partial characterization of an NADPH-dependent cytochrome P450 reductase from Liza klunzingeri liver (LkCPR). Its biochemical characterization revealed that LkCPR is a monomer with high activity in a broad range of pH (from 5 to pH 10) and temperature (from 40 to 70 • C) range, with pH 8 and 50 • C being the optimal conditions. LkCPR displayed lower K M values as well as a higher catalytic performance than other marine CPRs. Furthermore, LkCPR showed an unusually high stability at high temperatures and extreme pH values, the highest ones among the reported CPRs. Additionally, the enzyme exhibits the highest specific activity reported to date for a CPR.
Interestingly, some of the marine CPRs mentioned (namely, ScCPR [26] and LsCPR [27]) have been able to metabolize different substrates when reconstituted with CYPs. In particular, the system comprising both cytochrome P-450E and ScCPR was active on aromatic hydrocarbons [26]. The fact that LkCPR displays improved activity and higher temperature and pH stability when compared to its marine counterparts, leads us to believe that this enzyme could act as an optimal redox partner in a CYP-containing system.
In a more practical example, cytochrome CYP71B1 and NADPH cytochrome P450 reductase were immobilized through colloidal liquid aphrons (CLAs) [45]. Then, the obtained biocatalyst was successfully employed for the degradation of chlortoluron, an organic compound of phenylurea herbicides. Compared with the free enzyme, the immobilized biocatalyst displayed higher enzymatic and retained activity. This type of immobilized derivative arises as an interesting strategy for the development of robust catalysts for bioremediation. However, the harsh conditions often required for the immobilization process (alkaline conditions and long reaction times) could adversely affect the enzyme. In this sense, LkCPR could be a suitable candidate for the development of immobilized biocatalysts, since it exhibits remarkable stability and activity under extreme conditions.
To date, in the light of all the presented data, cytochrome P450 reductase from Liza klunzingeri arises as a suitable biocatalyst for the removal of marine pollutants.  Data Availability Statement: All data were included in the manuscript.

Conflicts of Interest:
The authors declare that they have no competing interest.