Chiang Mai Journal of Science

Selective Catalytic Reduction of 3-phenylpropiolonitrile to Cinnamonitrile by CtOYE: A Biocatalytic Approach to Partial Alkyne Reduction

1. INTRODUCTION

     Ene-reductases (ERs, EC 1.6.99.1) belong to the oxidoreductase family and catalyze the asymmetric reduction of activated alkenes [1, 2]. Old yellow enzymes (OYEs), a flavin-mononucleotide (FMN)-dependent subclass of ERs, are present in bacteria, fungi, and plants and catalyze the reduction of α,β-unsaturated ketones, aldehydes, nitroalkenes, carboxylic acids, and their derivatives [3-5]. In the presence of NAD(P)H, OYEs selectively reduce the C=C bond of α,β-unsaturated substrates to generate one or two new chiral centers. The canonical catalytic mechanism comprises an oxidative and a reductive half-reaction, where the tightly bound FMN on OYEs is reduced by NAD(P)H, the remaining NADP+ detaches from the active site, and the reduced FMNH2 is reoxidized by electron-deficient alkene substrate, delivering a hydride to the β-carbon of the substrate. Simultaneously, a conserved active-site tyrosine protonates the α-carbon to complete the reduction [6-11].
     Among ERs, the OYE family is the most extensively studied in biocatalysis, yet prior work has concentrated almost on alkene substrates, leaving alkyne reduction comparatively unexplored [12-14]. Alkynes can be partially reduced to alkenes or fully reduced to alkanes; controlling this chemoselectivity with traditional methods typically requires expensive metal catalysts and/or hazardous hydrogen gas and often affords poor stereoselectivity [15-17]. Therefore, the use of enzymatic methods for the selective reduction of alkynes is a promising alternative. In 2007, Müller et al. first showed that yeast-derived OYE1-3 completely reduced 4-phenyl-3-butyn-2-one to the corresponding alkane [12]. In 2021, Karrer et al. reported that the ER CaeEnR1—an ene-reductase of medium chain dehydrogenase/reductase (MDR) superfamily member from Cyclocybe aegerita—converted alkyne compounds, such as 3-phenylpropiolonitrile, mainly to the saturated product, albeit with modest conversion (< 45 %) [13]. More recently, González-Rodríguez et al. demonstrated that commercially available ene-reductases (EREDs) could reduce a broad range of alkyne nitriles, alkynones, alkynoates, and alkynals, giving predominantly the partially reduced alkene products [14].
     CtOYE is a newly characterized ER from Chroococcidiopsis thermalis [18], a spherical cyanobacterium renowned for thriving in extreme habitats—rock-soil biofilms, porous rock surfaces, desert pavements—and for its resistance to corrosion, freezing, short-wave UV, and ionizing radiation [5, 19-21]. Sequence analysis places CtOYE within the classical OYE family [6, 7], and earlier study has shown that it delivered high conversion and excellent enantioselectivity with alkene substrates such as α-alkyl-β-aryl enones and α,β-unsaturated esters, underscoring its industrial potential [18]. However, its ability to hydrogenate carbon-carbon triple bonds remains unexplored. Addressing this gap, we investigated the CtOYE-catalyzed reduction of 3-phenylpropiolonitrile, a representative alkyne substrate whose alkene and alkane products are key intermediates in pharmaceutical and fine-chemical synthesis [22, 23]. We combined product identification, molecular modeling (docking and dynamics simulations), and reaction optimization to elucidate the catalytic mechanism and maximize the conversion.

2. MATERIALS AND METHODS

2.1 Reagents
     3-Phenylpropiolonitrile, (E)-cinnamonitrile, and 3-phenylpropionitrile were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All other analytical-grade chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Plasmid extraction kits and BCA protein concentration assay kits were provided by Shanghai Tianhua Biotechnology Co., Ltd. (Shanghai, China). DNA polymerase and methylation digestion enzyme Dpn I were purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). Exnase II was provided by Vazyme Biotech Co., Ltd. (Nanjing, China).

2.2 Gene Cloning and Expression
     The codon-optimized gene encoding CtOYE (Chro_0590) was synthesized by Hangzhou Guannan Biotechnology Co., Ltd. (Hangzhou, China) and cloned into pET-28a(+) to yield the recombinant plasmid pET-28a-CtOYE, following our previously described protocol [24]. The recombinant pET28a-CtOYE was expressed in the Escherichia coli BL21(DE3). Recombinant E. coli BL21(DE3) carrying the plasmid pET28a-CtOYE was cultured in Luria-Bertani (LB) medium supplemented with 50 µg/mL kanamycin at 37 °C, 180 rpm for 12 h to obtain the seed liquid. The seed liquid was transferred to fresh LB medium containing 50 µg/mL kanamycin and grown under the same conditions until the OD₆₀₀ reached 0.6-0.8. Expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, and cultivation continued at 25 °C, 180 rpm for 12 h. Cells were harvested by centrifugation (8,000 × g, 4 °C, 10 min), resuspended in phosphate-buffered saline (PBS; 50 mM, pH 8.0), and disrupted by sonication for 10 min (3 s on, 4 s off) at 400 W. Insoluble debris was removed by centrifugation to obtain a clear soluble cell extract. The soluble cell extract was purified using a pre-packed Ni-NTA 6FF gravity column at 4 °C. The purified protein was desalted using a PBS buffer (50 mM, pH 8.0) by ultrafiltration and stored at -20°C for further use. Protein concentration was determined with a BCA Protein Assay Kit according to the manufacturer’s instructions. Purity and apparent molecular mass were assessed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE).

2.3 Enzyme Activity Assay
     The specific activity of CtOYE was assessed at 25 °C using a microplate reader by monitoring the decrease in absorbance of NADPH at 340 nm. The 200 μL reaction mixture contained 50 mM phosphate buffer (pH 9), 10 mM 3-phenylpropiolonitrile, 0.2 mM NADPH, and an appropriate amount of enzyme. One unit of enzyme activity (U) was defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute.

2.4 Biotransformation Reaction and Product Analysis
     In a standard reaction mixture, the components included 10 mM 3-phenylpropiolonitrile, 2.5 mg/mL CtOYE, 0.5 U/mL GDH, 20 mM glucose, 0.1 mM NADP⁺. The total reaction volume was 1 mL, and the reaction was carried out in a PBS buffer (50 mM, pH 8.0) at 30 °C and 180 rpm. After 12 h of reaction, the entire reaction mixture was extracted with ethyl acetate. The resulting extract was analyzed by gas chromatography (GC) using an Agilent 8890 GC system (Agilent, California, USA). The system was equipped with a flame ionization detection (FID) detector and a HP-5 capillary column (30 m length, 0.32 mm I.D., 0.25 μm film thickness, Agilent, California, USA). Nitrogen was used as the carrier gas at a flow rate of 1 mL/min with a split ratio of 1:100. The injector and detector temperatures were set at 250 °C. The column temperature program was as follows: initial temperature of 60 °C held for 11 min, then increased to 110 °C at 10 °C/min and held for 5 min, followed by an increase to 180 °C at 20 °C/min and held for 5 min. The injection volume was 1 µL. The configuration of the reduced product was identified by gas chromatography-mass spectrometry (GC-MS, Agilent 8890/7000D, California, USA) analysis of the standard sample of (E)-cinnamonitrile and the reaction sample.

2.5 Optimization of Reaction Conditions
     The reduction of 3-phenylpropiolonitrile using CtOYE was systematically optimized. The experimental variables were structured as follows: the reaction temperature was controlled within the range of 15-40 °C; the pH of PBS buffer (50 mM) was adjusted between 7.0 and 10.0. Other variables included glucose concentration (20-120 mM), GDH load (0.1-2.0 U/mL), NADP⁺ concentration (0.05-1.0 mM), and enzyme concentration (0.5-4 mg/mL). Reactions were carried out in a temperature-controlled shaker at 180 rpm for 12 h.

2.6 Molecular Docking
     Molecular docking was performed following the standard protocol of AutoDock Tools 1.5.6 [25-29]. The crystal structure of CtOYE (PDB ID: 6S32) was retrieved from the Protein Data Bank, and the ligand 3-phenylpropiolonitrile (CAS: 935-02-4) was obtained from PubChem. After preprocessing both receptor and ligand with AutoGrid4, docking calculations were carried out with AutoDock4 using a grid box centred at (45.121, 82.493, 42.755) and dimensions of (32.013, 65.618, 34.752). The results were visualized using PyMOL. The docking pose with low binding energy and a reasonable binding mode was selected for further analysis.

2.7 Molecular Dynamics (MD) Simulation
     MD simulations were conducted using GROMACS 2022.3 [30-33]. Small molecule preprocessing was performed using AmberTools22 to add General AMBER Force Field (GAFF), and Gaussian 16W was used to add hydrogen atoms and calculate Restrained Electrostatic Potential (RESP) charges, which were then incorporated into the topology files of the MD system. Simulations were performed under constant temperature (300 K) and pressure (1 bar) conditions, using the Amber99sb-ildn force field and TIP3P water model. An appropriate number of Na⁺ ions were added to neutralize the total charge of the system. The system was first energy-minimized using the steepest descent method, followed by 100,000 steps of NVT (constant number of particles, volume, and temperature) and NPT (constant number of particles, pressure, and temperature) equilibration with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, a free MD simulation was run for 5,000,000 steps with a time step of 2 fs, totaling 100 ns. After the simulation, trajectory analysis was performed using the built-in tools of the software to calculate the Root Mean Square Deviation (RMSD) [34], Radius of Gyration (RG) [35], and Solvent-Accessible Surface Area (SASA) [36].

3. RESULTS AND DISCUSSION

3.1 Expression, Purification, and Kinetic Analysis of CtOYE
     The CtOYE protein was heterologously expressed in E. coli BL21 (DE3), incorporating an N-terminal His6-tag. This poly-His tag facilitated the subsequent purification of the recombinant protein. Following expression, the cell extract containing CtOYE was subjected to purification via affinity chromatography using a Ni-NTA 6FF column. SDS-PAGE analysis of purified enzyme solution revealed a single band with an apparent molecular weight of approximately 45.1 kDa (Figure S1), confirming electrophoretic homogeneity and agreement with the theoretical molecular weight of CtOYE containing the poly-His tag. The preliminary characterization of CtOYE was performed by spectrophotometric determination based on the consumption of NADPH during the reduction of 3-phenylpropiolonitrile, with the results presented in Table 1 and Figure S2. The substrate affinity (K_m of 1.41 mM) and turnover number (kcat of 4.49 s⁻¹) were observed, resulting in a catalytic efficiency of 3.18 mM⁻¹ s⁻¹.

3.2 Identification of Reduction Product
     CtOYE requires the coenzyme NAD(P)H as a hydrogen donor for catalytic reduction of 3-phenylpropiolonitrile. However, the high cost of NAD(P)H necessitates cost-saving regeneration systems. We therefore employed a glucose/GDH co-substrate system to continuously regenerate NAD(P)H during reduction of 3-phenylpropiolonitrile (Figure 1). This approach provides economical reducing equivalents for enzyme catalyzed reduction reactions while minimizing NAD(P)H consumption [12]. The results showed that CtOYE effectively catalyzed the reduction of 3-phenylpropionitrile (Figure S3A), but not that of cinnamonitrile (Figure S3B), as evidenced by a comparison of their respective chromatograms with those of the standard samples (Fig. S3C-S3E). This absence of over-reduction aligns with the findings of González-Rodríguez et al. for similar alkyne nitriles using other EREDs [14]. To prove that the reduction process occurred due to the catalytic effect of CtOYE, appropriate control reactions were included. The results showed that when CtOYE was not present in the reaction system, the reactant peak was unchanged and the product was not detected (Figure S4A). Similarly, when GDH, NADP⁺ or glucose was not present in the reaction solution, the product peak was not detected (Figure S4B-S4D). The product peaks were detected only when CtOYE, GDH, NADP⁺, and glucose were added simultaneously (Figure S3A). The alkene product was identified as (Z)-cinnamonitrile by GC-MS analysis (Figure S5 and Figure S6). Molecular docking studies were carried out to explain the observed (Z)-selectivity (Figure 2). The substrate was located above the FMN in the active pocket of CtOYE and was roughly parallel to the isoalloxazine ring of FMN. This facilitated the formation of π-π interactions between the substrate and FMN, allowing the substrate to bind well to the active center. The binding energy of this docking pose was -6.72 kcal/mol, indicating that the binding of the substrate to CtOYE was relatively stable. The reaction mechanism of CtOYE-catalyzed carbon-carbon triple bond reduction was not completely consistent with that of carbon-carbon double bond reduction. The similarity was that the hydrogen required for β-carbon reduction of the substrate came from N5 of FMN, but the hydrogen required for α-carbon reduction was different. The canonical catalytic tyrosine residue Y183, which provided hydrogen for the substrate α-carbon during carbon-carbon double bond reduction, was relatively distant (4.5 Å) from the α-carbon of the alkyne substrate (Figure 2). In contrast, a non-catalytic tyrosine residue (Y351) was positioned closer (3.4 Å) to the α-carbon on the same face of the FMN isoalloxazine ring (Figure 2). This geometry is consistent with a mechanism where hydride transfer from FMNH₂ occurs to the β-carbon and protonation from Y351 occurs to the α-carbon, resulting in the syn addition of hydrogen and the formation of the (Z)-alkene product.

     To gain further insight into the differential reactivity of the alkyne and alkene, we performed MD simulations on both the CtOYE-3-phenylpropiolonitrile and CtOYE-cinnamonitrile complexes. Figure 3 showed the RG, SASA, and RMSD values for the two complexes. The RG and SASA values reflect the compactness of the protein molecule and the surface area exposed to the solvent, respectively. Smaller values indicate a more compact structure and weaker interactions with the solvent. RMSD measures the deviation of the protein structure from its initial conformation during the simulation. A lower RMSD value typically indicates that the molecular structure is more stable and undergo less change during the simulation. Initially, both complexes exhibited similar Rg and SASA values (Figure 3a, b). However, after ~40 ns, the CtOYE-cinnamonitrile complex showed significantly higher Rg (2.00 ± 0.01Å vs. 1.98 ± 0.01 Å) and SASA (160 ± 3 nm² vs. 152 ± 4 nm²), indicating reduced compactness and increased solvent exposure. This suggested that cinnamonitrile might not be as tightly bound into the CtOYE binding pocket, leading to weaker interactions with key catalytic residues and thus ineffective catalytic reactions. On the other hand, RMSD value was lower for CtOYE-cinnamonitrile (0.23 ± 0.01 Å vs.0.27 ± 0.01 Å) (Fig. 3c), suggesting overall structural rigidity. However, this rigidity might not be favorable for the catalytic reaction, because the catalytic process typically required a certain degree of structural flexibility to facilitate substrate binding, stabilization of the transition state, and release of the product. Therefore, the ability of CtOYE to catalyze the reaction of 3-phenylpropiolonitrile but not cinnamonitrile might be attributed to the more compact structure and higher dynamic flexibility of the CtOYE-3-phenylpropiolonitrile complex, allowing 3-phenylpropiolonitrile to better interact with the catalytic residues and stabilize the transition state. In contrast, although cinnamonitrile formed a stable complex with CtOYE, its structural characteristics were less favorable for the catalytic reaction, explaining the absence of over-reduction and the accumulation of alkene product.

3.3 Optimization of the Reaction Conditions for the Reduction of 3-phenylpropiolonitrile by CtOYE
     To maximize the catalytic efficiency of CtOYE in the reduction of 3-phenylpropiolonitrile, we systematically optimized pH, temperature, glucose concentration, NAD(P)⁺ concentration, GDH loading, and enzyme concentration. The results were presented and discussed in the following subsections.

3.3.1 Optimization of reaction pH
     pH profoundly influences enzymatic reactions by modulating both the conformational stability of the catalyst and the protonation states of its active-site residues, as well as the charge and structural integrity of the substrate. To identify the optimal pH for CtOYE-mediated reduction of 3-phenylpropiolonitrile, reactions were performed across the range 7.0-10.0. As shown in Fig. 4a, the conversion was relatively low at pH 7.0–8.0, gradually increased beyond pH 8.0, and peaked at 43.9 % at pH 9.0 before declining at higher pH values. This profile mirrors the report by the Robescu group, who observed maximal CtOYE activity at pH 9-10 when using 2-cyclohexen-1-one as the substrate [20]. The preference for alkaline conditions can be attributed to the optimal protonation state of key residues in the active center of CtOYE, which enhances substrate binding and promotes the catalytic reaction. Therefore, pH 9.0 was selected as the optimal condition for subsequent experiments.

3.3.2 Optimization of reaction temperature
     Temperature exerts a dual influence on enzymatic reactions: moderate elevations accelerate turnover by increasing the frequency of productive enzyme-substrate collisions, whereas excessive heat triggers irreversible denaturation that disrupts tertiary structure and obliterates active-site geometry. To identify the optimum temperature for CtOYE-mediated reduction of 3-phenylpropiolonitrile, we varied the temperature from 15 °C to 40 °C. As shown in Figure 4b, the conversion rose steadily with temperature and peaked at 52.6 % at 25 °C. However, when the temperature exceeded 30 °C, the conversion decreased. Therefore, 25 °C was established as the optimal reaction temperature.

3.3.3 Optimization of glucose concentration
     Glucose serves as the substrate for GDH driving the continuous regeneration of NAD(P)H from NAD(P)⁺ (Figure 1). NAD(P)H is essential for reducing the FMN cofactor of CtOYE (FMN to FMNH₂), which is the direct reductant for the substrate. Consequently, insufficient glucose limits cofactor regeneration and reaction progress. As shown in Fig. 4c, the conversion rose steadily with increasing glucose and peaked at 72.2 % when the glucose concentration reached 80 mM. Further increases in glucose concentration (up to 120 mM) yielded no significant improvement in conversion. This saturation behavior indicated that 80 mM glucose provided sufficient reducing equivalents (via NADPH regeneration) to sustain the catalytic cycle under the given conditions. Thus, 80 mM glucose was deemed optimal.

3.3.4 Optimization of NAD(P)⁺ concentration
     NAD(P)⁺ serves as the precursor to the obligate hydride donor NAD(P)H within the cofactor-recycling system (Figure 1). While NAD(P)H is consumed by CtOYE, the initial concentration of NAD(P)⁺ determines the total pool size of the cofactor available for recycling. Its cost necessitates optimization for process efficiency. As shown in Figure 4d, the conversion rose monotonically with increasing NADP⁺ from 0.05 mM to 0.2 mM, reaching a maximum of 72.2 % at 0.2 mM; further increases in NADP⁺ caused a slight decline. When NAD⁺ was substituted for NADP⁺, the profile was qualitatively similar, peaking at 0.2 mM with 51.1 % conversion, but overall yields remained consistently lower than those obtained with NADP⁺. This strong preference for NADP⁺ aligns with the kinetic characterization by the Robescu group, who reported CtOYE exhibits significantly higher affinity (lower K_m) and catalytic efficiency (k_cat/K_m) for NADPH compared to NADH (27-fold difference) using maleimide as a substrate [20]. This confirms NADPH is the preferred physiological coenzyme for CtOYE, applicable to both alkene and alkyne substrates. Therefore, 0.2 mM NADP⁺ was selected for further use.

3.3.5 Optimization of GDH loading
     GDH drives the regeneration of NADPH from NADP⁺ (Figure 1). A sufficient GDH loading is essential to ensure that the rate of NADPH formation matches the rate of its consumption by CtOYE, thereby preventing cofactor depletion from becoming rate-limiting. As illustrated in Figure 4e, the conversion rose with increasing GDH loading from 0.1 to 0.5 U/mL and plateaued at 72.2 % at 0.5 U/mL; further increases up to 2.0 U/mL afforded no additional improvement. This plateau indicated that 0.5 U/mL GDH was adequate to sustain the maximal turnover of CtOYE. Therefore, 0.5 U/mL GDH was chosen as optimal. 3.3.6 Optimization of enzyme concentration Enzyme concentration directly dictates the total number of active sites available for catalysis. Although higher concentration typically accelerate turnover, excessive concentrations can induce non-ideal effects—substrate depletion near the enzyme, elevated viscosity, protein aggregation, or steric hindrance—that ultimately diminish catalytic efficiency. As shown in Figure 4f, the conversion increased with CtOYE concentration from 0.5 to 2.5 mg/mL, peaking at 81.1 %. Beyond 2.5 mg/mL, the conversion declined slightly. The initial rise reflects the expected first-order dependence on active-site density under substrate-saturating conditions, whereas the subsequent decrease likely arises from enzyme aggregation, diffusion limitations, or steric hindrance at high protein densities, each of which reduces the effective concentration of accessible active sites. Therefore, 2.5 mg/mL was selected as the optimal enzyme concentration.

4. CONCLUSIONS

     This study established the ene-reductase from Chroococcidiopsis thermalis (CtOYE) as a novel biocatalyst that reduced 3-phenylpropiolonitrile exclusively to (Z)-cinnamonitrile, completely avoiding over-reduction to the alkane. The unprecedented (Z)-selectivity was attributed to a unique substrate-binding mode within the CtOYE active site, critically involving the non-canonical residue Y351. Systematic optimization of reaction parameters further elevated catalytic efficiency. Collectively, these results demonstrated CtOYE’s potential as a green and economically viable alternative to chemical methods for the selective synthesis of valuable (Z)-alkenes. CtOYE’s strict chemoselectivity renders it highly attractive for the late-stage functionalization of alkyne-bearing intermediates in pharmaceutical and agrochemical manufacturing, where such precision is difficult to achieve with traditional metal catalysts. Operating under mild and free of toxic reagents or precious metals, the enzyme can be readily integrated into multi-step chemoenzymatic cascades or continuous-flow systems, streamlining the synthesis of fine chemicals and complex active pharmaceutical ingredients while minimizing downstream purification. Future investigations should focus on expanding the substrate scope assessment of CtOYE, particularly for structurally diverse alkynes, employing protein engineering to enhance catalytic efficiency and robustness for industrial applications, and addressing key limitations of the current in vitro system, namely cofactor cost and enzyme stability. Promising strategies include rational/semi-rational protein engineering of enzyme, developing whole-cell biocatalysts, and implementing enzyme immobilization technology to improve catalyst recyclability.

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