Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower kcat/KM value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.


Materials
The aldehydes 3a-d used as starting materials, as well the other organic, inorganic reactants and solvents used in the chemical synthesis of the substrates were purchased from Sigma-Aldrich (St.
Louis, MO, USA) and/or Alfa-Aesar (Haverhill, MA, USA).All solvents were purified and dried by standard methods as required.The primers used for the mutagenesis were purchased through the services of Genomed (Debrecen, Hungary).IPTG, Phusion Hot Start DNA Polymerase, dNTPs, DpnI, XhoI, Bpu1102I, agarose were all products of Thermo Fischer Scientific (Waltham, MA, USA).Plasmid extraction kit and ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA).LB medium were from Liofilchem (Roseto, Italy), protease inhibitor cocktail from Hoffman La-Roche (Basel, Switzerland), while the Ni-NTA Superflow resin used for affinity chromatography was from Qiagen (Hilden, Germany).

Instrumentation
The 1 H and 13 C NMR spectra were recorded on Bruker (Billerica, MA, USA) Avance spectrometers operating at 400 MHz and 101 MHz / 600 MHz and 151 MHz, respectively.Spectra were recorded at 25 °C in CDCl 3 , D 2 O, MeOD, DMSO. 1 H and 13 C NMR spectra were referenced internally to the solvent signal.MS spectra and LC-MS analysis were recorded on Agilent 6410 Triple Quadrupole LC/MS mass spectrometry system.LC-MS measurements of rac-1a-d were performed using Phenomenex Kynetex 2.6 µm C18 100Å, 50×2.1mmcolumn, acetonitrile 70%, water (0.1 HCOOH) 30% as mobile phase at 0.3 ml/min flow rate.The MS detector was operated in positive electrospray ionization mode, with source temp 35 °C, capillary voltage 4000 V, fragmentor 120 V and with MS2Scan mode, at least +/-50 amu around molecular ion.High performance liquid chromatography (HPLC) analyses were conducted with an Agilent (Santa Clara, CA, USA) 1200 instrument.Kinetic measurements were performed on and Agilent 8453 UV-Vis spectrophotometer.For the PCR reactions, the Mastercyler from Eppendorf (Hamburg, Germany) was used.Gene sequencing services were performed through Genomed (Debrecen, Hungary).Protein purification with size exclusion chromatography were performed with an Äkta purifier FPLC (GE Healthcare, Sweden).Preparative scale enzymatic reactions were performed with Heidolph (Schwabach, Germany) Titramax 1100 equipped with incubator module.DNA concentrations were measured with Thermo Fischer Scientific (Waltham, MA, USA) Nanodrop 2000 instrument.SDS-PAGE was performed using the BioRad (Hercules, CA, USA) Mini-Protean Tetra Cell vertical electrophoresis unit, using 10% Tris-glycine Laemmli-gels.

Chemical synthesis of substrates rac-1a-d and 2a-d
Starting from the commercially available aldehydes 3a-d acrylic esters 4a-d were obtained through Wittig reaction, using the corresponding triphenyl-phosphoranylidine.The reduction of esters 4a-d with DIBAL-H afforded the primary alcohol derivatives 5a-d, which through an oxidation with manganese-dioxide, followed by another Wittig reaction with triphenylphosphoranylidene were converted into styrylacrylic esters 7a-d.Mild alkaline hydrolysis from esters 7a-d resulted in the styrylacrylates 2a-d (Scheme S1).The styrylic alcohols 5a-d, obtained as described above (Scheme S1) were converted into the diethyl acetamidomalonates 9a-d via malonic acid coupling of the brominated compounds 8a-d.
Further, through a mild alkaline hydrolysis of 9a-d, followed by the subsequent decarboxylation of 10a-d, the N-acylated amino acids rac-11a-d were obtained.Finally, deprotection of rac-11a-d afforded the racemic amino acids rac-1a-d (Scheme S2).

Synthesis of styrylacrylic esters 4a-d
Into the stirred solution of aldehydes 3a-d (18 mmol) in dry toluene (60 mL) triphenylphosphoranylidine (8.1 g, 23.4 mmol) was added in portions.The mixture was stirred at 110 °C until completion of the reaction (approx.12 h, checked by TLC), followed by the evaporation of the solvent.The residue was purified by silica gel column chromatography using CH 2 Cl 2 as eluent leaving the product 4a-d as colorless or yellow viscous oil in 85-90% yield.The 1 H, 13 C-NMR spectra of the products were in accordance with the reported data.
The mixture was stirred for 1 h at 0 °C and after completion of reaction (checked by TLC), the excess of DIBAL-H was quenched with 1 mL of MeOH, followed by addition of 2 mL of saturated solution of NH 4 Cl.The mixture was stirred again at 0 °C for 15 min, followed by extraction with CH 2 Cl 2 (3 × 25 mL) and water (3 × 25 mL).The organic phase was dried over anhydrous MgSO 4 , filtered and concentrated in vacuum.The residue was purified by silica gel column chromatography using CH 2 Cl 2 as eluent leaving the product 5a-d as colorless or yellow viscous oil in 90-95% yields.The 1 H-, 13 C-NMR spectra of the products are in accordance with the reported data. 2

Synthesis of the styrylacroleins 6a-d
Into the stirred solution of alcohols 5a-d (3.7 mmol) in dry CH 2 Cl 2 (25 mL) was added manganesedioxide (1.6 g, 18.5 mmol) in portions.The mixture was stirred for 24 h until the reaction completed (checked by TLC).The mixture was filtered under vacuum through a short silicagel pad, using CH 2 Cl 2 as eluent, followed by the removal of the solvent in vacuum to give the product 6a-d as yellow or orange viscous oil in 84-89% yield.The 1 H, 13 C-NMR spectra of the products are in accordance with the reported data. 3

Synthesis of the styrylacrylates 2a-d
The reactions from aldehydes 6a-d were performed similarly as described in Section 3.1.1.for the reactions from 3a-d (Synthesis of acrylate esters 4a-d).The products were purified by silica gel column chromatography using CH 2 Cl 2 as eluent leaving the styrylacrylic esters 7a-d as yelloworange oils in 81-85% yields.
The esters 7a-d were subsequently hydrolyzed by the addition of 10% NaOH (5 mL) and stirring under reflux until the reaction completed (4-20 h, checked by TLC).The resulted mixture was diluted with water (5 mL), and extracted with ethyl acetate (3×5 mL).The aqueous phase was acidified with 10% HCl solution to pH 1-2, followed by extraction with ethyl acetate (3 × 10 mL).
The combined organic phases were dried over anhydrous MgSO 4 , filtered, followed by the evaporation of the solvent in vacuum to give 2a-d as white or yellow-white solid in 70-76% yield.

Synthesis of racemic amino acids rac-1a-d (Scheme S2)
Into the stirred solution of styryl allylalcohol 5a-d (10 mmol) in dry CH 2 Cl 2 (50 mL) triphenylphosphine (5.5 g, 21 mmol) was added, followed by a portionwise addition of N-bromosuccininimide (3.56 g, 20 mmol).The mixture was stirred for at room temperature, until the reaction was completed (0.5-2 h, checked by TLC).The solvent was evaporated in vacuum, followed by the purification of the residue by silica gel column chromatography using CH 2 Cl 2 as eluent leaving the bromides 8a-d which were directly used in the coupling reaction with malonic acid.
NaH (0.48 g, 11 mmol, 55% suspension in mineral oil) was added to dry N,N-dimethylformamide (20 mL) and stirred at room temperature under argon atmosphere.After 30 min, diethyl acetamidomalonate (2.23 g, 10 mmol) was added and the mixture was stirred for 30 min.To the ice-cooled mixture was added dropwise a solution of the bromide 8a-d (10 mmol) dissolved in dry N,N-dimethylformamide (10 mL).The reaction mixture was stirred at room temperature for 3 h and at 60 °C for 4 h.The solution was cooled and poured on a water-ice mixture (200 mL).
The formed precipitate was filtered off and dried under reduced pressure.The product was redissolved in CH 2 Cl 2 (1-2 mL) and purified by silica gel column chromatography using CH 2 Cl 2 as eluent leaving the diethyl 2-acetamido-2-cinnamyl malonic acid derivatives 9a-d.
The obtained malonic acid 9a-d (6 mmol) was dissolved in methanol (5 mL), followed by addition of 10% NaOH solution (5 mL).The resulted mixture was stirred at room temperature for 3 h, and at 60 °C until the reaction completed (2-3 h, checked by TLC).Subsequently, the methanol was removed in vacuum and the concentrated aqueous solution was diluted with water (10 mL) and extracted with ethyl acetate (3 × 10 mL).The pH of the aqueous phase was adjusted to 1 with 10% HCl solution, followed by extraction with ethyl acetate (3 × 15 mL).The combined organic phases were dried over Na 2 SO 4 , filtered, followed by the removal of the solvent in vacuum.
The formed dicarboxylic acid derivative 10a-d was suspended in toluene (10 mL) and heated under reflux for 20 h.The solvent was removed in vacuum to leave the products 11a-d.
The N-acetyl styrylalanine 11a-d was suspended in dry 1,4-dioxane (10 mL) followed by the addition of concentrated HCl (0.5 mL).The resulted mixture was heated under reflux until the reaction completed (4-12 h, checked by TLC).The organic solvent was evaporated in vacuum and the obtained precipitate was washed with anhydrous diethyl ether (3 × 5 mL) to give the pure racemic styrylalanines rac-1a-d.

Michaelis-Menten curves with non-linear fitting for calculation of the kinetic parameters (KM, vmax, kcat)
The kinetic measurements were based on UV-spectroscopy by monitoring the production of the acrylic derivative 2a-d at wavelengths where the corresponding amino acids rac-1a-d showed no absorption The values of v max and K M were obtained from non-linear regression fitting of the Michaelis-Menten curves by MATLAB using the equation ).The k cat values were calculated according to equation where [E]-represents the enzyme concentration used in the kinetic assay (μM) -Table S2.
Table S2.Enzyme concentrations used in the kinetic assays                 , measurements performed at 20 °C, wavelength used for UV detection: 260 nm.

Chiral HPLC analysis of the final products from wt-PcPAL-catalyzed kinetic resolutions of rac-1a-d
Samples were taken from the enzymatic reactions after different time intervals as described in the main manuscript (Experimental part; ammonia elimination reactions from rac-1a-d) followed by the HPLC analysis using the chiral separation method indicated above.
The peaks appearing at R t = 2.9-3.2min, represent the elution front, which contain the produced acrylic derivatives 2a-d.In order to rule out that the asymmetrical peak form is caused by the presence of impurities, besides the LC-MS analysis of rac-1a-d (see Supporting information, chapter 3.2), which supports the high purity level of the compounds, we also analyzed commercially available L-styrylalanine L-1a using the developed chiral method.The obtained asymmetrical peak, similar with those obtained from the enzymatic reactions or from the separation of rac-1a-d supports that the peak shape of the chiral HPLC chromatograms may be due to variable binding poses to the chiral stationary phase.

Investigation of the ammonia addition onto styrylacrylates 2a-d by the PcPAL variants
The enzymatic ammonia addition reactions were performed in triplicate as follows: The 6M NH 3 -solutions (1 mL, pH 10, adjusted with CO 2 ), containing styrylacrylates 2a-d at a final concentration of 5 mM, and purified PcPAL (50 µg of F137V-, F137A-, F137G-, or wt-PcPAL), were incubated at 30 °C with shaking at 250 rpm.Conversions were monitored using reverse phase high performance liquid chromatography (HPLC) as described earlier in section 5.1.Samples (30 μL) were taken from the reaction mixtures after 24 h time intervals, quenched by adding an equal volume of MeOH, vortexed and centrifuged (13400 rpm, 12100 g, 2 min).The supernatant was used directly for HPLC analysis after transferring through a 0.22 μm nylon membrane filter.Despite monitoring the reactions over 20 days, no product (L-1a-d) formation could be detected by HPLC.In order to rule out enzyme deactivation during the 20 days reaction monitoring, fresh batch of enzyme was added to the mixture after each 48 h period.

Molecular modeling principles
Besides the practical work, we tried to rationalize the experimental data with additional theoretical work.Our research philosophy comprised of a straightforward, physics-based approach.We aimed to acquire the ligand pose/conformation sets of the important biocatalytic sequence members and corresponding energy values in silico.These computational data were used to approximate changes on the potential energy surface and thus characterize certain phenomena of the catalysis.Furthermore, the enumeration of the possible ligand conformations could reveal energetically preferred, but unproductive states which can slow down or block the catalytic process.
Following this principle, three major properties were calculated.One was the ligand binding energy ( b ΔE), calculated with a modified MM-GBSA methodology, appreciated as one of the most useful and meaningful theoretical quantities, from the standpoint of experimental work.To approximate the Gibbs energy of activation, two descriptors were used.One was associated with the acidity of the β-proton ( cB ΔE), and the other one was associated with the steric strain ( Ster ΔE).
Moreover, these energy differences were transformed relative to the same property of L-Phe or (E)-cinnamate with wt-PcPAL that makes these values not just more intelligible, but enables us to compare intermediate states with different connectivity with molecular mechanics.9However, one should be warned that the absolute values of these calculated properties should not be compared directly to experimental values, and should be even scaled in some cases.
For such investigations an atomistic model of the enzyme -representing its structure at the assay conditions, with special attention paid to the active site -and knowledge about the reaction mechanism is needed.Unfortunately, the only crystal structure determined for PcPAL so far did not contain a substrate or substrate analogue. 7Moreover, the essential Y110containing loop in this structure was not in the active conformation. 8Thus, a structure with a catalytically active loop conformation required partial homology modeling 8,10 and refinement of the Y110-loop region.
Next, the enzyme-substrate complexes within the active site were constructed.Committing to these assumptions, the corresponding covalent N-MIO and preceding substrate-binding, noncovalent intermediate states of all studied substrates were created introducing our induced-fit covalent docking protocol which was previously applied in the case of another MIO enzyme, Pantoea agglomerans PAM. 9 This protocol enumerates the possible ligand poses in the active site while accounting for the conformational change of the enzyme too.Moreover, explicit

Molecular modeling methods
For molecular modeling studies, the tetrameric partial homology model of PcPAL 10 was used as a starting structure.The crude model was completed and adjusted using the Protein Preparation Wizard 11 in two steps: i) hydrogen atoms were added and bond orders were assigned, and ii) the hydrogen bond network, tautomeric states, side chain conformations of selected amino acids, and ionization states were determined and optimized corresponding to the experimental assay conditions.In all four active centers, Y110 was set deprotonated and Y351 protonated.Protein pKa were predicted using PROPKA. 12Next, residues 103-126 of the catalytically essential Y110 containing loop and further residues within 3 Å distance were subjected to loop modeling.This step consisted of two repetitions of side chain prediction of the previously mentioned residues with C α -C β vectoring incorporated, thus simulating also the small scale movement of the backbone (implicit water solvation model: VSGB, number of steps:2) using Prime. 13The final model was minimized with Prime 13 [RMSG: 0.1 kcal mol -1 Å -1 , algorithm: TNCG, implicit water solvation model: VSGB).
The refined and completed structure served as a starting point to create an overall protein model corresponding to the experimental assay conditions.The buffer solution solvated model was created by the Desmond program suite. 14The buffer solvated model was then equilibrated with a slightly modified default equilibration protocol, applying harmonic constraints to the Cartesian coordinates of buried hydrogen atoms and all protein heavy atoms.A spherical model of the active site with a radius of 27 Å, centered on the exocyclic methylene carbon of the MIO prosthetic group of chain C, was cut off and capped with acetyl and N-methylamino groups.
N-MIO type covalent complexes of substrates rac-1a-d and 2a-d were constructed by our induced-fit covalent docking protocol.This involved the creation of initial conformations of compounds by docking with Glide 15 into a modified and artificially enlarged active site in which the residues L134, F137, L138, L256, V259, and I460 were exchanged to Ala residues, further the MIO prosthetic group was reduced to Ala + Gly and three water molecules in the active site were removed.
After having docked into the enlarged active site, all side chains and the MIO group were restored.Residue 137 was in all cases changed from Ala to the residue corresponding to the actual enzyme mutant.For compounds D-and L-1a-d, a covalent bond between the nitrogen atom of the amino group and the exocyclic carbon of MIO was created, for compounds 2a-d, the MIO group was restored as an aminated NH 2 -MIO group, and Y110 was set protonated.The ligands and the residues in close proximity were minimized, and finally, redundant conformations were eliminated with MacroModel. 16After this step, side chain conformations of residues L134, F137, L138, L256, V259, I460, and N260 were predicted with Prime (with C α -C β vectoring, implicit water solvation model: VSGB, number of steps:2).Next, water molecules in the active center and its immediate vicinity were predicted and placed with Grand Canonical Monte Carlo simulation using Desmond. 14Finally, a minimization of all ligands and the same set of atoms in the 6 Å proximity resulted in the final models and energies using Prime.The OPLS3 force field was applied in all molecular mechanics calculations and simulations.
Binding energy values ( b ΔE) were calculated with a modified MM-GBSA methodology.The standard MM-GBSA method involves the minimization, incorporating also implicit solvation, of the ligand-receptor complex, followed by the subsequent minimization of the receptor and the ligand individually after separation.Finally, the MM-GBSA score ( b ΔE) is calculated according to Eq. S1, where the three terms are the final energies of the previously mentioned minimizations, respectively, and X137 refers to the F137V mutant (X=V) or wild-type (X=F) PcPAL enzyme.Our modifications involved i) the substitution of the ligand energy term in Eq.S1 with a value obtained after mixed Monte Carlo/low-mode conformational search with MacroModel 16 [solvation model: VSGB, number of steps: 2000, energy window: 31 kJ mol -1 ] and a reoptimization with Prime [used with the same settings as before], ii) the substitution of the receptor energy term in Eq.S1 uniformly for all ligands with the corresponding apoenzyme structure's energy, and iii) the solvation of the N-MIO intermediate complexes were done with explicit solvation rather than implicit solvation with the aid of Grand Canonical Monte Carlo simulation using Desmond. 14This mixed use of implicit and explicit solvation is undesired in (S1) general, however the application of implicit solvation to the minimization of the complexes introduces unacceptably large errors, and in addition, the favorable cancellation of errors made the mixed scheme convenient to provide relevant molecular structures and energies.
Values of the descriptor Ster ΔE were calculated according to Eq. S1, with the small change that the term E ligand was obtained with minimization without the application of any solvation model.
Values of descriptor cB R ΔE were calculated according to Eq. S2, where L-Phe;wt-PcPAL in the subscript refers to the same quantity calculated with L-Phe in the wt-PcPAL active center, E cB is the single point energy of the model of the conjugated base, and E N-MIO is the single point energy of the N-MIO model, both calculated at the B3LYP/6-311+G(dp) level with the GD3 dispersion correction model using the program suite MRCC (www.mrcc.hu). 17 For the N-MIO model, the previously described enzyme-substrate models were used such as the atoms of the enzyme were deleted, and the dangling substrate-MIO bond was capped with a methyl group.For the conjugated base model, the pro-S β-proton was removed from the N-MIO model, and the remaining one was adjusted to help the β-carbon of the alanyl substructure to form sp 2 hybrid state arrangement.
Quantities Ster ΔE and b ΔE (denoted here as Y in general) were also evaluated relative to the corresponding values of L-Phe or (E)-cinnamate with wt-PcPAL throughout the text, according to Eq.S3.
F137X Y ΔE has the purpose to incorporate the change when the mutation F137V is introduced, and it is calculated according to Eq. S4.
(S2) (S3) (S4) temperature was taken to be 303 K.In a few cases, multiple psc and/or pst conformations were obtained (3a with wt-and F137V-PcPAL, 4a with F137V-PcPAL).In these cases, only the two lowest energy active conformations were taken into account.Strictly speaking, they were two psc conformations in the case of 3a with F137V-PcPAL, but they were used in the non-linear estimation nevertheless.Data in Table 4. of the main text show the exact lowest energy psc and pst conformations of 3a with F137V-PcPAL.
Descriptor values and molar fractions were obtained for 4-fluoro-, 4-trifluoromethyl-, 4-bromo, and 4-cyanostyrylalanine with the same procedure as discussed earlier.Predicted k cat values (k cat,pred ) were determined according to Eq. S12.Statistical analysis was conducted with the use of Statistica. 18

Figure S1 .
Figure S1.Non-linear fitting curves for the ammonia elimination from L-Phe catalyzed by wt-PcPAL; measured in triplicate.

Figure S2 .
Figure S2.Non-linear fitting curves for the ammonia elimination from L-Phe catalyzed by F137V-PcPAL; measured in triplicate.

Figure S3 .
Figure S3.Non-linear fitting curves for the ammonia elimination from L-Phe catalyzed by F137A-PcPAL; measured in triplicate.

Figure S4 .
Figure S4.Non-linear fitting curves for the ammonia elimination from L-Phe catalyzed by F137G-PcPAL; measured in triplicate.

Figure S5 .
Figure S5.Non-linear fitting curves for the ammonia elimination from L-1a catalyzed by wt-PcPAL; measured in triplicate.

Figure S6 .
Figure S6.Non-linear fitting curves for the ammonia elimination from L-1a catalyzed by F137V-PcPAL, measured in triplicate.

Figure S7 .
Figure S7.Non-linear fitting curves for the ammonia elimination from L-1a catalyzed by F137A-PcPAL; measured in triplicate.

Figure S8 .
Figure S8.Non-linear fitting curves for the ammonia elimination from L-1a catalyzed by F137G-PcPAL; measured in triplicate.

Figure S9 .
Figure S9.Non-linear fitting curves for the ammonia elimination from rac-1a catalyzed by wt-PcPAL; measured in triplicate.

Figure S11 .
Figure S11.Non-linear fitting curves for the ammonia elimination from rac-1b catalyzed by wt-PcPAL; measured in triplicate.

Figure S13 .
Figure S13.Non-linear fitting curves for the ammonia elimination from rac-1c catalyzed by wt-PcPAL; measured in triplicate.

Figure S15 .
Figure S15.Non-linear fitting curves for the ammonia elimination from rac-1d catalyzed by wt-PcPAL; measured in triplicate

5. 1 .
Determination of the conversion by HPLC In order to determine the conversion in the PcPAL-catalyzed enzymatic transformations, the relative response factor of the acrylic derivatives 2a-d compared to the amino acids rac-1a-d was determined by injecting the mixture of known composition of the corresponding amino acid rac-1a-d and the corresponding acrylic derivative 2a-d onto an Agilent Zorbax Eclipse XDB-C8 column (150 × 4.6 mm; 5 µm).

Table S1 .
The wavelengths used for monitoring the production of acrylic derivatives 2a-d and molar extinction coefficients (ε) at the corresponding wavelengths

Table S3 .
HPLC methods and response factors, used for the conversion value determinations * Mobile phase: A: NH 4 OH buffer (0.1 M, pH 9.0) / B: MeOH; flow rate: 1.0 mL min -1

Table S4 .
Retention times of the enantiomers of rac-1a-d obtained on Chiralpak Zwix (+) chiral column solvation of the complexes was performed with the aid of Grand canonical Monte Carlo simulations.Lastly, the final, refined enzyme-substrate complexes were energetically evaluated with Schrödinger's Prime function.Docking did not indicate significant, energetically preferred unproductive states with any substrate and PcPAL mutant combinations in the case of the substrate-binding, non-covalent intermediate directly leading to the subsequent covalent N-MIO