Improving Theaflavin-3,3′-digallate Production Efficiency Optimization by Transition State Conformation of Polyphenol Oxidase

Theaflavins (TFs) are good for health because of their bioactivities. Enzymatic synthesis of TFs has garnered much attention; however, the source and activity of the enzymes needed limit their wide application. In this study, a microbial polyphenol oxidase from Bacillus megaterium was screened for the synthesis of theaflavin-3,3′-digallate (TFDG). Based on structural and mechanistic analyses of the enzyme, the O-O bond dissociation was identified as the rate-determining step. To address this issue, a transition state (TS) conformation optimization strategy was adopted to stabilize the spatial conformation of the O-O bond dissociation, which improved the catalytic efficiency of tyrosinase. Under the optimum transformation conditions of pH 4.0, temperature 25 °C, (−)-epigallocatechin gallate/epicatechin gallate molar ratio of 2:1, and time of 30 min, Mu4 (BmTyrV218A/R209S) produced 960.36 mg/L TFDG with a 44.22% conversion rate, which was 6.35-fold higher than that of the wild type. Thus, the method established has great potential in the synthesis of TFDG and other TFs.

The production of TFDG involves direct extraction, chemical oxidation, or enzymatic methods [9]. However, the direct extraction is not suitable for industrial-scale production because of high costs, wastage of resources, and a low extraction rate. The chemical oxidation synthesis method has disadvantages such as a low extraction rate of TFs and a complicated operational process. Furthermore, according to the European and United States legislation, chemically synthesized additives cannot be used in cosmetics, foods, or beverages [10]. Consequently, the enzymatic synthesis of TFDG has garnered increasing attention, which is a natural synthesis method and can greatly decrease the production cost [11].
Polyphenol oxidase (PPO) and peroxidases (PODs) are two groups of enzymes that are important for the synthesis of TFs. Zhang et al. [12] integrated transcriptome and

Structure and Catalytic Mechanism of BmTyr
BmTyr structure was obtained from the PDB database (PDB:3nm8) (Figure 2a), which was similar to that of the type 3 copper protein family containing two Cu (II) ions. These two Cu(II) ions were coordinated by six conserved histidine residues, which serve as the major cofactors. We aimed to identify the complex structure of BmTyr with Cu (II) ions and a ligand (EGCG); however, only the apo-BmTyr structure was obtained with 2.56 Å resolution (PDB ID:8HPI) ( Figure S2). Therefore, based on 3nm8, a reactive binding mode M1 was created ( Figure 2b). M1 contained two copper ions that were 3.5 Å apart and two bridging solvent molecules that were designated as oxygens. Furthermore, an MD analysis of M1 was performed, and the conformation with the highest evaluation rate was obtained through cluster analysis (Figure 2c). This conformation was named model 2 (M2), which was used for subsequent docking experiments with EGCG. The docking experiments produced a theoretically complex structure of M2-EGCG, which was named M3 (Figures 2d and S3).

Structure and Catalytic Mechanism of BmTyr
BmTyr structure was obtained from the PDB database (PDB:3nm8) (Figure 2a), which was similar to that of the type 3 copper protein family containing two Cu (II) ions. These two Cu(II) ions were coordinated by six conserved histidine residues, which serve as the major cofactors. We aimed to identify the complex structure of BmTyr with Cu (II) ions and a ligand (EGCG); however, only the apo-BmTyr structure was obtained with 2.56 Å resolution (PDB ID:8HPI) ( Figure S2). Therefore, based on 3nm8, a reactive binding mode M1 was created ( Figure 2b). M1 contained two copper ions that were 3.5 Å apart and two bridging solvent molecules that were designated as oxygens. Furthermore, an MD analysis of M1 was performed, and the conformation with the highest evaluation rate was obtained through cluster analysis ( Figure 2c). This conformation was named model 2 (M2), which was used for subsequent docking experiments with EGCG. The docking experiments produced a theoretically complex structure of M2-EGCG, which was named M3 (Figures 2d and S3).
The docking results are shown in Figure 3a. The flexibility of R209 facilitated the stabilization of EGCG in the BmTyr binding pocket. F197, a bulky residue, showed a weak π-π interaction with the aromatic ring of EGCG, forming a specific gate that prevented a substrate with incorrect orientation from entering the pocket (Figure 3b). The conserved water molecules, which were stabilized by E195 and N205, acted as proton donors and receptors during the process of hydroxylation. The other residues, including M184, P201, G216, V217, and V218, interacted to fix EGCG to facilitate the reaction (Figure 3b).
Based on the docking results and the reported mechanism underlying PPO-mediated reactions, a hypothetical catalytic mechanism of BmTyr was inferred (Figure 3c) [17][18][19][20]. This reaction contained the following steps: (i) phenolic -O11H bond formation with the bridging oxygen atom (TS1), (ii) O-O bond dissociation of the bridging oxygen species (TS2), (iii) another phenolic -O9H bond formation, and (iv) proton transfer from the catechol to the bridging oxygen group accompanied by electron transfer to afford the quinone product. A detailed analysis of the energy profile showed that the rate determination step is the O-O bond dissociation, regardless of the active copper center state [21]. Similar to the catechol oxidase diphenolase cycle, three protons are required to enter the stage where the O-O bond is cleaved [22]. The O-O bond cleavage was a prohibitively large barrier for this reaction. MD simulations showed that the distance between the -O11H of the EGCG and the O1 of the bridging oxygen atom of the active center was 4.66 ± 0.57 Å ( Figure S8), which made the hydride transfer and proton transfer difficult. Thus, optimizing this critical step required the optimum conformation of TS2. The docking results are shown in Figure 3a. The flexibility of R209 facilitated the stabilization of EGCG in the BmTyr binding pocket. F197, a bulky residue, showed a weak π-π interaction with the aromatic ring of EGCG, forming a specific gate that prevented a substrate with incorrect orientation from entering the pocket (Figure 3b). The conserved water molecules, which were stabilized by E195 and N205, acted as proton donors and receptors during the process of hydroxylation. The other residues, including M184, P201, G216, V217, and V218, interacted to fix EGCG to facilitate the reaction (Figure 3b).
Based on the docking results and the reported mechanism underlying PPO-mediated reactions, a hypothetical catalytic mechanism of BmTyr was inferred (Figure 3c) [17][18][19][20]. This reaction contained the following steps: (i) phenolic -O11H bond formation with the bridging oxygen atom (TS1), (ii) O-O bond dissociation of the bridging oxygen species (TS2), (iii) another phenolic -O9H bond formation, and (iv) proton transfer from the catechol to the bridging oxygen group accompanied by electron transfer to afford the quinone product. A detailed analysis of the energy profile showed that the rate determination step is the O-O bond dissociation, regardless of the active copper center state [21]. Similar to the catechol oxidase diphenolase cycle, three protons are required to enter the stage where the O-O bond is cleaved [22]. The O-O bond cleavage was a prohibitively large barrier for this reaction. MD simulations showed that the distance between the -O11H of the EGCG and the O1 of the bridging oxygen atom of the active center was 4.66 ± 0.57 Å (Figures S8), which made the hydride transfer and proton transfer difficult. Thus, optimizing this critical step required the optimum conformation of TS2.

Improving BmTyr Catalytic Efficiency via TS2 Conformation Optimization
To determine the optimum conformation of TS2, restrained MD simulations were conducted. First, the MD state model was constructed by defining geometric criteria on the basis of the existing M3 and previous studies on the mechanism [20]. During the restrained MD calculations, the coordinates of the active site atoms were fixed. Subsequently, stable conformations were selected by cluster analysis to construct the active site cluster model, namely the TS2 state model ( Figure S5). A total of 15 residues (M61, E158, M184, E195, F197, P201, N205, R209, Q214, M215, G216, V217, V218, A221, and F227) within 5 Å of the TS2 were selected for the subsequent Rosetta enzyme design (Figure 4a).
During Rosetta enzyme design, to accommodate the large backbone changes introduced at each step, we used PSSMs to limit amino acid choices to identities that were common in the multiple-sequence alignment ( Figure S6) [23]. Thus, 119 mutants were designed in this study. Computational mutation scanning was performed to predict the effects of each mutation (∆∆G). Mutants with scores lower than that of the WT at the same residue position were selected as hits, which created 22 variants for experimental valida-

Improving BmTyr Catalytic Efficiency via TS2 Conformation Optimization
To determine the optimum conformation of TS2, restrained MD simulations were conducted. First, the MD state model was constructed by defining geometric criteria on the basis of the existing M3 and previous studies on the mechanism [20]. During the restrained MD calculations, the coordinates of the active site atoms were fixed. Subsequently, stable conformations were selected by cluster analysis to construct the active site cluster model, namely the TS2 state model ( Figure S5). A total of 15 residues (M61, E158, M184, E195, F197, P201, N205, R209, Q214, M215, G216, V217, V218, A221, and F227) within 5 Å of the TS2 were selected for the subsequent Rosetta enzyme design (Figure 4a).
During Rosetta enzyme design, to accommodate the large backbone changes introduced at each step, we used PSSMs to limit amino acid choices to identities that were common in the multiple-sequence alignment ( Figure S6) [23]. Thus, 119 mutants were The 22 variants were expressed in E. coli BL21 (DE3), which were purified and tested for TFDG production ( Figure S7). Compared with the TFDG titers of WT, 19 of the 22 variants showed improved TFDG titers, which confirmed the effectiveness of this method. Among the 19 improved variants, Mu 1 (BmTyr R209S ), Mu 2 (BmTyr V217L ), and Mu 3 (BmTyr V218A ) showed an almost two-fold increase in TFDG titer; Mu 3 , the most efficient mutant, showed a 2.43-fold increase in TFDG titer (438.19 mg/L) compared with that of WT. To further increase this activity, R209S and V217L were combined with V218A, resulting in the variants Mu 4 (BmTyr V218A/R209S ) and Mu 5 (BmTyr V218A/V217L ). Mu 4 showed the highest TFDG titer (675.30 mg/L), corresponding to a 4.29-fold increase compared with that of the WT (Figure 4b). The kinetic parameters of WT and its variants are shown in Table 1. Compared with WT, Mu 4 showed increased activity toward ECG and EGCG. The specific activities of Mu 4 toward ECG and EGCG, which were 51.14 and 86.58 U/mg, respectively, were 3.68and 4.67-fold higher than that of the WT. The V max /K m of Mu 4 toward ECG and EGCG were 14.59 U and 27.17 U min −1 mg −1 mM −1 , respectively, which were 6.05-and 6.97-fold higher than that of the WT.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 15 designed in this study. Computational mutation scanning was performed to predict the effects of each mutation (ΔΔG). Mutants with scores lower than that of the WT at the same residue position were selected as hits, which created 22 variants for experimental validation ( Figure S7). Using PSSMs and ΔΔG ≤ 0 R.e.u. as the cutoff, severely destabilizing mutations were eliminated. The 22 variants were expressed in E. coli BL21 (DE3), which were purified and tested for TFDG production ( Figure S7). Compared with the TFDG titers of WT, 19 of the 22 variants showed improved TFDG titers, which confirmed the effectiveness of this method. Among the 19 improved variants, Mu1 (BmTyr R209S ), Mu2 (BmTyr V217L ), and Mu3 (BmTyr V218A ) showed an almost two-fold increase in TFDG titer; Mu3, the most efficient mutant, showed a 2.43-fold increase in TFDG titer (438.19 mg/L) compared with that of WT. To further increase this activity, R209S and V217L were combined with V218A, resulting in the variants Mu4 (BmTyr V218A/R209S ) and Mu5 (BmTyr V218A/V217L ). Mu4 showed the highest TFDG titer (675.30 mg/L), corresponding to a 4.29-fold increase compared with that of the WT (Figure 4b). The kinetic parameters of WT and its variants are shown in Table 1. Compared with WT, Mu4 showed increased activity toward ECG and EGCG. The specific activities of Mu4 toward ECG and EGCG, which were 51.14 and 86.58 U/mg, respectively, were 3.68-and 4.67-fold higher than that of the WT. The Vmax/Km of Mu4 toward ECG and EGCG were 14.59 U and 27.17 U min −1 mg −1 mM −1 , respectively, which were 6.05and 6.97-fold higher than that of the WT.
To elucidate the mechanism underlying the increased enzymatic activity, MD simulations were performed on the Mu4-EGCG complex. Compared with BmTyr-EGCG, the hydride transfer distance of the Mu4-EGCG complex was shortened from 4.66 ± 0.57 to 3.02 ± 0.31 Å (Figures 5a,b and S8). This suggests that the distance between the -O11H of the EGCG and the O1 of the bridging oxygen atom of the active center was shortened in Mu4. Furthermore, V218A and R209S eliminated steric hindrance (Figure 5c,d). The side chain residues of valine and arginine in Mu4 were larger than that of the WT, which increased the steric hindrance of the transition state. Providing more space for the substrate, where the catalytic residues are present, is conducive to the stability of the transition state, which may enable the release of the products.  The data represent mean ± s.d., as determined from three independent experiments.
To elucidate the mechanism underlying the increased enzymatic activity, MD simulations were performed on the Mu 4 -EGCG complex. Compared with BmTyr-EGCG, the hydride transfer distance of the Mu 4 -EGCG complex was shortened from 4.66 ± 0.57 to 3.02 ± 0.31 Å (Figures 5a,b and S8). This suggests that the distance between the -O11H of the EGCG and the O1 of the bridging oxygen atom of the active center was shortened in Mu 4 . Furthermore, V218A and R209S eliminated steric hindrance (Figure 5c,d). The side chain residues of valine and arginine in Mu 4 were larger than that of the WT, which increased the steric hindrance of the transition state. Providing more space for the substrate, where the catalytic residues are present, is conducive to the stability of the transition state, which may enable the release of the products.

Discussion and Conclusions
In this study, the efficacy of PPO from different sources in TFDG synthesis was compared, and BmTyr, with a high conversion rate and wide application value, was screened. To improve the conversion of TFDG, we performed mechanism analysis and computational design to optimize the TS2 conformation. TFDG was effectively synthesized using ECG and EGCG as substrates and the optimum mutants as biocatalysts, increasing the conversion of TFDG by 6.35-fold. Therefore, the established method has practical value for TFDG synthesis and great potential for the synthesis of other TFs.
A stable enzyme source-supporting method, using microbiological fermentation, was selected. To date, most enzyme sources related to in vitro catalytic synthesis of TFDG were derived from plant extracts, which are mainly concentrated in natural PPOs that are directly isolated from tea leaves, mushrooms, or pear fruits [13,24]. Plant PPOs are not conducive to the industrialization of tea pigment production because it requires a large number of tea leaves to meet the application requirements. Furthermore, there were large differences in

Discussion and Conclusions
In this study, the efficacy of PPO from different sources in TFDG synthesis was compared, and BmTyr, with a high conversion rate and wide application value, was screened. To improve the conversion of TFDG, we performed mechanism analysis and computational design to optimize the TS2 conformation. TFDG was effectively synthesized using ECG and EGCG as substrates and the optimum mutants as biocatalysts, increasing the conversion of TFDG by 6.35-fold. Therefore, the established method has practical value for TFDG synthesis and great potential for the synthesis of other TFs.
A stable enzyme source-supporting method, using microbiological fermentation, was selected. To date, most enzyme sources related to in vitro catalytic synthesis of TFDG were derived from plant extracts, which are mainly concentrated in natural PPOs that are directly isolated from tea leaves, mushrooms, or pear fruits [13,24]. Plant PPOs are not conducive to the industrialization of tea pigment production because it requires a large number of tea leaves to meet the application requirements. Furthermore, there were large differences in the enzyme composition of plants in different seasons, and their effects on the synthesis of TFs were inconsistent. The large-scale production of TFs requires a stable enzyme source. We selected seven functionally known microbial PPOs from the NCBI database via data mining. The recombinant strain BmTyr was selected to improve the biological preparation of TFDG because it has the advantages of a simple culture method, abundant raw material sources, high enzyme yield, and good enzyme production stability.
A transition-state conformation optimization strategy was adopted, which provided an effective tool for obtaining beneficial mutations in BmTyr to improve TF synthesis. Many strategies have been adopted to engineer proteins and to improve their properties. For example, Zhou et al. [25] obtained activating mutations from BmTyr after screening almost 20,000 colonies for random mutations. This approach required enormous screening efforts. In this study, BmTyr was efficiently engineered on the basis of the Rosetta enzyme design for the computer simulation of stable TS to improve oxidation efficiency. A total of 19 beneficial mutants were obtained from the designed library (22 mutants) that produced TFDG. Compared with the traditional random mutation, this strategy effectively reduces the workload. In a different study, theaflavins were synthesized by extracting polyphenol oxidase from eggplants. The results showed that the total content of theaflavins could reach 40.7 mg under optimum reaction conditions [26]. Ngure, F. M. et al. [27] used PPO from tea to oxidize catechins, and the final theaflavin content reached 26.99 µmol/g. PPOs from snow lotus fruits have also been used to oxidize catechins to form theaflavins. The final estimate of theaflavins in the reaction system was 65 µM dry weight/h [28]. Under the catalysis of the optimum mutant BmTyr V218A/R209S , the titer of TFDG reached 960.36 mg/L with 44.22% conversion, which was 6.35-fold higher than that of the WT. These results showed the effectiveness of our strategy and may be useful for improving the properties of other enzymes.
Despite the abovementioned results, several factors restrict the production of TFs. Although the TFDG generation rate is high, the industrial application of free enzymes is impeded because they are either inactivated or cannot be recovered for reuse. Immobilized enzymes can effectively overcome the limitations of free enzymes. A suitable carrier should be found to immobilize PPO, which can make the enzyme reaction continuous and automatic, reduce production costs, and improve efficiency. Our ongoing study will address these issues and improve the utilization of BmTyr.

Strains, Plasmids, and Chemicals
All plasmids and strains are listed in Table S1, and their genetic constructions are shown in the supporting information. Commercial solvents, standards, and reagents were purchased from Meryer Chemicals (Shanghai, China), Sigma Aldrich (Shanghai, China), J&K Chemicals (Beijing, China), TCI Chemicals (Shanghai, China), and Aladdin Reagents (Shanghai, China) and used without further purification.

Culture and Purification
All desired purified proteins and mutants were obtained under the following operating conditions. Strains were first grown in LB medium with suitable antibiotics at 37 • C and 200 rpm. When the OD 600 of the culture broth reached 0.6-0.8, IPTG was added to make a final concentration of 0.1 mg/mL. The cells were inducted at 16 • C for 16 h, collected by centrifugation (5000 rpm, 10 min), suspended in lysis buffer (20 mM Tris-HCl (pH 8.0), 20 mM imidazole, 500 mM NaCl) and then disrupted using a high-pressure homogenizer. The resulting crude lysate was centrifuged at 10,000 rpm at 4 • C for 30 min, and the protein containing a His-tag was trapped on Ni-NTA Superflow resin for 30 min. Subsequently, the elution buffer (20 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 0.5 M imidazole) was used to release the protein. The protein concentration was determined using a NanoDrop 2000c spectrophotometer (Thermo Scientific). The purity of the proteins was determined by SDS-PAGE.

Construction and Screening
The seven samples of PPOs obtained by genome mining were overexpressed in E. coli via pET28a and purified to obtain pure enzymes that were used to catalyze the conversion of ECG and EGCG to TFDG. This 2 mL screening system contained 0.2 M MacIlvaine buffer, 5 mM substrate, 0.1 mM Cu 2+ , and 0.06 mM pure enzymes at pH 5.0, 30 • C, and 200 rpm.

HPLC Analysis
The titer of TFDG was determined using high-performance liquid chromatography (HPLC). The HPLC analytical conditions were as follows: XBridge ® -C18 column (5 µm, 4.6 × 250 mm), column temperature of 30 • C, detection wavelength of 280 nm, injection quantity of 10 µL, and the flow rate was 1.0 mL/min. Chromatography was performed with an elution of 30% A for 5 min, 34% A for 3 min, and finally, 30% A for 2 min, where mobile phase A was 100% acetonitrile and mobile phase B was 2% acetic acid aqueous solution.
The conversion was calculated as follows: where a is the amount of TFDG synthesized, and b is the amount of ECG added to the system. The conversion values of the reaction catalyzed for TFDG synthesis were calculated.

Protein Crystallization and Structure Determination
Crystals of apo-BmTyr were obtained after 3 days at 20 • C in 96-well plates, in which 0.8 µL protein solution (25 mg/mL) and an equal volume of reservoir solution (0.1 M zinc acetate dehydrate, 0.1 M sodium acetate trihydrate; pH 4.6; 12% w/v PEG4000) were mixed, as per the optimized conditions for crystallization. All crystals were flash frozen with 25% glycerol as an antifreezing solution in liquid nitrogen. X-ray diffraction data were collected using a Bruker D8 QUEST diffractometer, and the data sets were indexed, integrated, and merged using XDS [29]. The structure of BmTyr was solved using the molecular replacement, with the protein structure of 3nm8 as the search model. Iterative cycles of model building and refinement were performed in Coot [30] and Refmac [31] programs. The data collection and refinement statistics of the apo-BmTyr crystal structure are listed in Table S4. Structural figures were prepared using PyMOL v2.3.3 (Schrödinger, LLC, New York, NY, USA).

Initial Structural Preparation
The initial structure of BmTyr was obtained using Bacillus megaterium as a template (PDB ID:3nm8 [32]). Based on 3nm8, a reactive binding mode, namely BmTyr (M1), was created. The protonation states of the charged residues were determined at a constant pH of 5.0 on the basis of the pKa calculations using H ++ [33] while taking into account the local hydrogen bonding network. The His residues were assigned as HID. Asp and Glu residues were deprotonated, whereas Lys and Arg were protonated. The prepared protein was neutralized by adding Na + ions and solvated into a truncated octahedral TIP3P [34] water box with a 10 Å buffer distance on each side. The M1 system consisted of 27,473 atoms. The system was equilibrated through a series of minimization steps that was interspersed by short molecular dynamics (MD) simulations, during which the restraints on the protein backbone heavy atoms were released gradually (with a force constant of 10, 2, 0.1, and 0 kcal/mol·Å 2 ). The system was gradually heated up to 303 K in 50 ps in which harmonic potentials were used to positionally restrain the protein backbone heavy atoms (with a force constant of 10 kcal/mol·Å 2 ). Finally, the standard unrestrained MD simulation with periodic boundary condition at 303 K and 1 atm was carried out for up to 100 ns. Based on the dynamically stable BmTyr (M2) binary complex structure determined by MD simulations, EGCG was docked into the active center to obtain the possible binding mode of EGCG (BmTyr-EGCG, M3). A representative pre-equilibrated structure of BmTy-EGCG was used as a template to create the starting coordinates of Mu 4 -EGCG by changing the two mutated residues (V218A/R209S). To check for statistical convergence, each simulation was carried out in triplicate with different initial atomic velocities that were randomly generated according to the Maxwell-Boltzmann distribution.

Molecular Docking
In the equilibrium trajectories of the M2 binary complex, 2000 snapshots were selected and divided into ten groups through a hierarchical agglomerative (bottom-up) approach [35]. EGCG was fully optimized at the B3LYP/6-31G(d) level using the Gaussian 16 [36] package, and docked into the active site of each group representative snapshot. Molecular docking was performed by the Lamarckian genetic algorithm local search method in AutoDock 4.2 and AutoDockTools-1.5.6 [37]. The docking approach was employed on a rigid receptor conformation. One-hundred independent docking runs were undertaken. Reasonable conformations were then selected as the binding conformations for MD simulations.

MD Simulations
The pre-equilibrated M3 structures and possible catalytically reactive binding modes of EGCG were used as the starting conformations for the MD simulations of the proteinligand complexes. The partial charges of EGCG were fitted with HF/6-31G(d) calculations, and the restrained electrostatic potential (RESP) [38,39] protocol was implemented by the Antechamber module in the Amber 16 package [40]. Each system was initially neutralized with Na + counter ions and solvated with TIP3P [34] water in a truncated octahedron box at a 10 Å buffer distance. The resulting systems M2, M3, and Mu 4 -EGCG contained 27,473, 27,188, and 27,230 atoms, respectively. Next, each system was equilibrated with a series of minimization that was interspersed by short MD simulations, during which the restraints on the protein backbone heavy atoms were released gradually (with force constants of 10, 2, 0.1, and 0 kcal/mol·Å 2 ). The system was then gradually heated from 0 to 303 K for 50 ps, in which a 10 kcal/(mol·Å 2 ) restraint was applied to heavy atoms on the protein backbone. Finally, an extensive MD simulation of 50 ns was performed at constant temperature and pressure. The pressure was maintained at 1 atm and coupled with isotropic position scaling. The temperature was controlled at 303 K using the Berendsen thermostat method [41]. Long-range electrostatic interactions were treated using the particle mesh Ewald (PME) method [42], and a 12 Å cutoff was applied to both PME and van der Waals (vdW) interactions. A time step of 2 fs was employed along with the SHAKE algorithm [43] for hydrogen, and periodic boundary conditions were used. The atomic positions were stored every 2 ps for further analysis. Each system was checked for stability (structure, energy, and temperature fluctuations) and convergence (root mean square deviations (RMSD) of the structures).

Rosetta Design
Active-site positions were selected for design, and at each position, position-specific scoring matrices (PSSMs) were used to constrain amino acid choices to identities. We generated a sequence alignment, and the less favorable amino acids were removed by natural selection. We then used Rosetta computational mutation scanning. Each "allowed" mutation was modeled in the context of the WT structure, and the energy difference (∆∆G) between the WT and the mutant was calculated. We defined the space of "conformation optimization" mutations as mutations with ∆∆G ≤ 0 Rosetta energy units (R.e.u.), generating many designs for experimental testing.

Directed Evolution Experiments
Directed evolution experiments were performed by whole-plasmid polymerase chain reaction (PCR) using KOD-Plus-Neo (TOYOBO). The primers used are listed in Table S2. The resulting PCR products were digested with DpnI to remove the template plasmid. Furthermore, 10 µL of digested products was transformed into E. coli BL21 (DE3) cells for subsequent screening or DNA sequencing (GENEWIZ, China). These mutants were cultured into LB medium and shaken at 37 • C for 12 h. Mutants were then diluted 1:10 into 150 mL fresh Terrific Broth (TB) medium. After shaking at 37 • C for 3 h (for cell growth), the temperature was decreased to 25 • C for 16 h (for protein expression). Cells were then collected by centrifugation.

Activity Assay
The activities of BmTyr and the mutants were spectrophotometrically measured using ECG and EGCG as substrates [44,45]. The activity was determined by measuring the initial rate of quinone formation by an enzyme-labeled instrument under the following conditions: 5 mM substrate, 0.6 mM enzymatic solution containing 0.1 mM Cu 2+ , 200 mM Mcilvaine Buffer (pH 5.0) in a final volume of 200 µL, at 200 rpm and 30 • C for 5 min. Finally, the reaction was terminated by adding 50 µL of 2 M trichloroacetic acid and centrifuging the solution at 12,000× g for 5 min. The absorbance of the samples was analyzed at 405 nm, and a reaction mixture without the enzymatic solution was used as a negative control. One unit of enzymatic activity was defined as the amount of enzyme that changes the absorbance by 0.01/min.

Kinetic Assay
The kinetic parameters of the enzymes were determined by measuring the initial rates of product formation at different concentrations of the substrate (EGCG:0-16 g/L, and ECG:0-8 g/L) for 5 min. Other assay conditions were the same as those described for the corresponding activity assays. The samples were withdrawn, extracted, and analyzed using HPLC. The K m and V max values were calculated using nonlinear regression according to the Michaelis-Menten equation in the Origin software.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28093831/s1, Table S1. Strains and plasmids used in this study. Table S2. The primers for gene amplification. Table S3. The primers for the saturated mutation. Table S4. Data collection and refinement statistics. Figure S1. The HPLC chromatograms of TFDG reaction products. Figure S2. The whole structure of BmTyr (PDB ID:8HPI). Figure S3. The construction process of the whole theoretical complex structure. Figure S4. RMSD and RMSF of BmTyr WT and BmTyr V218A/R209S . Figure S5. The whole TS2 state model. Figure S6. Multiple sequence alignment of BmTyr with homologous tyrosinase sequences from superfamily. Figure S7. Redesign of BmTyr. Figure S8. The distance between the -O11H of the hydroxy oxygen atom of EGCG and the O1 of the bridging oxygen atom of the active center.