Influence of the aromatic moiety in α- and β-arylalanines on their biotransformation with phenylalanine 2,3-aminomutase from Pantoea agglomerans

Biocatalysis and Biotransformation Resea Cluj-Napoca, Arany János str. 11, RO-4 paizs@chem.ubbcluj.ro Department of Organic Chemistry and Techn and Economics, M} uegyetem rkp. 3, H-1111 B bme.hu Agricultural Institute, Centre of Agricult Sciences, Brunszvik u. 2, H-2462 Martonvás Institute of Organic Chemistry, Karlsruhe In Allee, D-76128 Karlsruhe, Germany SynBiocat Ltd, Lázár deák u 4/1, H-1173 B † This paper is dedicated to Professor Alb 90 birthday. ‡ Electronic supplementary informa 10.1039/c6ra02964g Cite this: RSC Adv., 2016, 6, 56412


Introduction
Nowadays there is an ever increasing demand for optically pure b-amino acids mainly by the pharmaceutical industry and peptide syntheses. 1 In particular, the biological characteristics of the b-amino acids, along with their use as precursors of various heterocycles and as chiral auxiliaries in enantioselective syntheses have aroused lively interest in their chemistry. This induced rapid development of synthetic procedures for the preparation of enantiopure b-amino acids and their congeners. 2 Until recently most of the biocatalytic approaches for them relied on kinetic resolution with hydrolytic enzymes, such as lipases, 2 acylases 3 and hydantoinases. 4 An attractive method for the synthesis of enantiopure, nonnatural b-amino acids is based on the use of phenylalanine 2,3-aminomutase (PAM). According to their stereochemical preference there are two kinds of phenylalanine 2,3-aminomutases (PAMs), the one of plant origin (EC 5.4.3.10) is producing (R)-b-phenylalanine, 5 while the other one is of bacterial origin (EC 5.4.3.11) and is leading to (S)-b-phenylalanine. 6,7 Both are members of the class I lyase-like family also including tyrosine 2,3-aminomutase (TAM, EC 5.4.3.6), 8 phenylalanine ammonia-lyase (PAL, EC 4.3.1.24 and EC 4.3.1.25), 9 tyrosine ammonia-lyase (TAL, EC 4.3.1.23 and EC 4.3.1.25), 10 and histidine ammonia-lyase (HAL, EC 4.3.1. 3). 11 All of them utilize the same protein-derived prosthetic group, 3,5dihydro-5-methylidene-4H-imidazol-4-one (MIO) (Fig. 1A), formed autocatalytically in the active site from an XSG motif which is typically Ala-Ser-Gly. 11 Less frequently MIO could be formed from a Thr-Ser-Gly (in PaPAM and in SmPAM), a Ser-Ser-Gly (in HAL from Fusobacterium nucleatum) or a Cys-Ser-Gly (in HAL from Streptomyces griseus) motif as well. 12 The broad range of aromatic and heteroaromatic amino acids tolerated as substrates by these enzymes was also exploited for the preparation of a wide range of non-natural aryl and heteroaryl aand b-amino acids. 13 PAM from Taxus canadensis (TcPAM) forming (R)-b-phenylalanine was used for the partial biotransformation of (S)-aphenylalanine and its derivatives into their (R)-b-isomer, 5,14 while the closely related Taxus chinensis (TchPAM) as biocatalyst was employed in the enantioselective ammonia addition to (E)cinnamate producing a mixture of enantiopure (S)-a-and (R)-bphenylalanine. 15 In a later study, signicant shi of the regioisomeric preference towards the b-isomers was achieved by site directed mutagenesis. 16 Wanninayake et al. have exploited some unnatural amino acids as amino group donors. 17 The amino group of these substrates was transferred by TcPAM intermolecularly to another arylacrylate skeleton to form mixtures of aand b-arylalanines.
Acting on a-phenylalanines the (S)-isomer-preferring PAM (EC 5.4.3.11) forms (S)-b-phenylalanine transposing exclusively the amino group of the (S)-a-phenylalanine to give (S)-bphenylalanine (Fig. 1B). 6 Among the known members of the PAM family producing (S)-b-phenylalanine are AdmH from Pantoea agglomerans (PaPAM) 6 and EncP from Streptomyces maritimus (SmPAM). 7 These enzymes have important applications in the preparation of the antibiotic andrimid, 18 further various chiral phenylalanine derivatives 19 as well as in the synthesis of the anticancer drug Taxol. 20 The crystal structure of PaPAM complexed with phenylalanine to its active site, supported a reaction mechanism proceeding through two N-MIO containing intermediates. 6 This was also in agreement with QM/MM calculations on TAL 21 and PAL 22 supporting ammonia elimination via an N-MIO intermediate and suggesting the formation of similar N-MIO complexes as a common feature of the mechanism for all MIOenzymes. 21 According to these propositions, the steps starting from either aor b-phenylalanine are quite similar such as: (i) formation of a covalent enzyme-substrate complex via Michael addition of the amino group of the substrate onto MIO, (ii) ammonia elimination from the covalent N-MIO intermediate resulting in a cinnamate binding intermediate state (Fig. 1A). The mechanism proceeds further with (iii) ammonia readdition and (iv) the release of the product. Occasionally, cinnamic acid can appear as a by-product (Fig. 1A), supposedly due to intermittent opening of the Tyr78-containig loop resulting in a leak from the main cycle at the cinnamate binding intermediate state.
SmPAM, described earlier as a lyase, has been shown lately to be closely related to PaPAM (63% overall sequence identity and 76% sequence similarity). 7 More recently, Weise et al. investigated SmPAM in the context of ammonia addition to several aryl-substituted (E)-cinnamic acid analogues. 23 They found that SmPAM converted a range of arylacrylates to a mixture of (S)-aand (S)-b-arylalanines. The enzyme exhibited variable regioselectivity, much affected by ring substituents, but introduction of certain active site mutations could shi regioselectivity in either direction. However, in SmPAM-catalysed isomerization of (S)-aand (S)-b-arylalanines the enantioselectivity was incomplete in many cases. 23 Substrate specicity of PaPAM was tested with a wide range of aromatic and heteroaromatic (S)-a-arylalanines. 19 Electronic and steric effects of substituents at the aromatic ring signicantly inuenced both catalytic efficiency and the formation of arylacrylates as by-products. It was observed that 3-substituted (S)-a-phenylalanines were transformed faster than the 2-or 4substituted isomers. In order to explain these observations, computational analysis of substrate-PaPAM structural interactions was performed by substrate docking studies. 19 Recently it was shown that recombinant whole cell E. coli expressing PaPAM could also produce enantiopure (S)-b-arylalanines from (S)-a-arylalanines. 24 Worth noting, that PaPAM failed to catalyse the transformation of several 2-substituted (S)-a-phenylalanines studied. 19,24 Results and discussion The time course of the reaction (ESI ‡ material) showed that aer a relatively short time period (2-6 hours) an equilibrium state is reached in case of both enzymatic reactions, using rac-bphenylalanines (Fig. 1C) or rac-a-phenylalanines (Fig. 1B) as substrates. Therefore the results aer 20 h show the nal product distribution near to the equilibrium state, aer longer reaction times further product formation was not observed.
Results with the (AE)-a-arylalanines (rac-1a-l) demonstrated that mutase and/or lyase activity of PaPAM was much affected by the nature of the aromatic moiety of the substrates. Importantly, high enantiopurity of the products [>98% ee for (S)-2a-f, h, i, k, l] indicated high enantioselectivity of the PaPAMcatalysed isomerization in the a / b-direction. The observed high stereoselectivity of the PaPAM-catalysed isomerization of the (S)-a-and (S)-b-arylalanines was a major advantage compared to the incomplete stereoselectivity of SmPAMcatalysed isomerizations. 23

Transformation of (AE)-b-arylalanines (rac-2a-l) with PaPAM
In order to explore the potential of kinetic resolutions of (AE)-aand b-arylalanines for the preparation of antipodal products, we extended our study to the reactions of (AE)-b-arylalanines rac-2al ( Fig. 2B and Table 2). The similar time course proles of the product formation in PaPAM catalyzed reactions from (S)-bphenylalanine and rac-b-phenylalanine (ESI ‡ material) supported that the unreactive (R)-b-phenylalanine did not act as a signicant inhibitor. In contrast to the (AE)-2-substituted aphenylalanines with large substituents (rac-1g, j), which were apparently not accepted as substrates by PaPAM, all the (AE)-2substituted b-phenylalanines in the present study (rac-1d, g, j) were smoothly transformed. On the other hand, sluggish or no conversion was observed with PaPAM using as substrates (AE)-3and (AE)-4-substituted b-phenylalanines bearing bulky electron withdrawing substituents (rac-2c, h, i, k, l).
Effects of pH and ammonia concentration on the PaPAMcatalysed isomerization of (AE)-a-and b-(thiophen-2-yl)alanine (rac-1b and rac-2b)c Prompted by the fact that conversions from (AE)-a-and (AE)-b-(thiophen-2-yl)alanine (rac-1b and rac-2b) with PaPAM were similar to those from the natural substrates i.e. (AE)-a-and bphenylalanine (rac-1a and rac-2a) but more of the by-product [(E)-3-(thiophen-2-yl)acrylate, 3b] was formed (Tables 1 and 2), transformations of these substrates were studied in more detail by varying pH or ammonia concentration. An alteration of pH of the buffer solution in the range of 7-9 [at 100 mM (NH 4 ) 2 CO 3 ] was indifferent to conversion (data not shown), unlike changing the (NH 4 ) 2 CO 3 concentration in the range of 50-1000 mM (at pH 8) which signicantly inuenced product compositions ( Table 3).

Substrate (Ar)
x c a ee > 98% when not stated otherwise. b No reaction was observed. c Not observed. d x 1 , x (S)-2 and x 3 represent the relative molar fractions of the reaction components as determined by 1 H-, 19 F-NMR measurements.  In molecular mechanics the bonded terms measure the strain compared to a hypothetical zero-point energy. Thus potential energies derived from molecular mechanics calculations cannot be compared directly if the molecular structures to be related do not have exactly the same atom connectivity. In this modelling study it was assumed that for all investigated compounds the common alanine part of aand b-arylalanines underwent in the corresponding N-MIO intermediates similar structural changes 6,21 (Fig. 1). Therefore, it was possible to compare the difference of the energies calculated for the N-MIO  (Table 4).
Statistical analysis of the experimental and computational data revealed that compounds (S)-1, 2a-i, k, l could be classied Table 3 Composition of the reaction mixtures obtained from (AE)-aor (AE)-b-(thiophen-2-yl)alanine (rac-1b or rac-2b)   Energy difference c (kcal mol À1 ) a Difference of conversions is dened as: (1 À x 2 ) À (1 À x 1 ), where x 1 , x 2 are the corresponding molar fraction values listed in Tables 1 and 2 Table 4) based on the difference of conversions [dened as: (1 À x 2 ) À (1 À x 1 ), where x 1 , x 2 are the corresponding molar fraction values listed in Tables 1 and 2]. The three categories: A: (1 À x 2 ) À (1 À x 1 ) < À0.09; E: À0.09 # (1 À x 2 ) À (1 À x 1 ) # 0.09 and B: (1 À x 2 ) À (1 À x 1 ) > 0.09 could be correlated with the regioisomeric preferences (Table 4, Fig. 4). One-way ANOVA testtreating the categories (Table 4) as the independent and the conversion differences (Tables 1 and 2) as the dependent variablesindicated signicant difference between categories A and B at the a ¼ 0.050 level. In addition, further nonparametric testshaving lower statistical power than parametric methods but without the requirement of normal distribution of datawere also performed. The Kruskal-Wallis ANOVA and Mann-Whitney U tests resulted only somewhat higher p values (0.100 and 0.053, respectively) than the threshold. Moreover, the median test showed also signicant differences between the categories. Overall, the statistical tests supported the nding that categories A and B are truly separate. The computational study extended with statistical analysis revealed that the cases when one of the regioisomers was converted much faster (or is the only one to be converted) the energetics of the regioisomeric N-MIO-type enzyme-substrate complexes was one of the most important factors governing the outcome of the reaction. In the case of the intermediate group (category E) involving small aromatic moieties contribution of the steric effects are much less pronounced.
The computational results listed in Table 4 suggest that the reactions of 2-substituted a-phenylalanines [(S)-1d, g and (S)-2d, g: category B in Table 4 and Fig. 4] are strongly disfavoured because the energy calculated for the a-N-MIO intermediates [(S)-1d, g N-MIO ] is much lower than that calculated for the corresponding b-N-MIO intermediates [(S)-2d, g N-MIO ]. This was in full agreement with experimental observations indicating slow or no reaction from the (AE)-2-substituted a-phenylalanines (rac-1d, g, j) with PaPAM, in contrast to high conversions from 2-substituted (AE)-b-phenylalanines (rac-2d, g, j) under the same conditions. The situation for the 3-and 4-substituted phenylalanines with bulky substituents [(S)-1c, h, i, k, l and (S)-2c, h, i, k, l: category A in Table 4 and Fig. 4] was the opposite. In good correlation with the experimental regioisomeric preferences, much lower energies were calculated for the b-N-MIO intermediates [(S)-2c, h, i, k, l N-MIO ] than for the corresponding a-N-MIO intermediates [(S)-1c, h, i, k, l N-MIO ]. This could explain sluggish or no conversion of barylalanines containing bulky substituents at positions 3 or 4 (rac-2c, h, i, k, l) with PaPAM and much higher conversions of the a compounds (rac-1c, h, i, k, l) under the same conditions. In addition, the energy differences corresponding to the direction of reaction with higher conversions and assuming a disallowed ip of the aromatic ring (Table 4, values in parentheses) showed remarkable differences compared to the sterically non-restricted values and the hindered ip of the aromatic ring further ampli-ed the difference in reactivity mentioned before. Because of the hindered ip, in case of certain regioisomeric amino acid pairs the aromatic ring in a given N-MIO intermediate adopts different conformation depending on whether the actual N-MIO intermediate is at the substrate side [step (ii) in our postulated mechanism, Fig. 1A] or at the product side [step (iii) in our postulated mechanism, Fig. 1A].

Conclusion
According to our results, regioselectivity and activity of PaPAM towards aand b-arylalanines are mainly inuenced by the nature of the aromatic moiety of the substrates. PaPAM catalyses the synthesis of the corresponding (S)-b-enantiomer from racemic 3and 4-substituted a-phenylalanines more smoothly than that starting from racemic 2-substituted a-phenylalanines, the latter being poor or no substrates of the enzyme. Contrarily, racemic 2substituted b-phenylalanines were good substrates and provided the (S)-a-enantiomers smoothly while the racemic 3-or 4substituted b-phenylalanines were almost no substrates. Importantly, in all but one case (for racemic 2-nitro-b-phenylalanine), the isomerizations were stereospecic giving mixture of unreacted (R)-a-or (R)-b-arylalanines with an enantiomeric excess depending on conversion, and enantiopure (S)-a-or b-arylalanines as products, along with various amounts of arylacrylate as by-product. Computational and statistical analysis revealed signicant correlation between the energetics of the N-MIO intermediate states forming from the (S)-a-or b-arylalanines and the regioisomeric preferences of PaPAM in case of substrates with bulky electron withdrawing substituents on the aromatic ring. In several cases "hysteresis" was postulated: the conformation (thus energy) of a given regioisomeric N-MIO intermediate formed from the given regioisomeric substrate differed from that conformation which resulted in the given regioisomer as product.

Reagents and analytical methods
The starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Carl Roth (Karlsruhe, Germany) and used without purication. Solvents were puried and dried by standard methods. The racemic amino acids rac-1a-l rac-2a-l and the a,b-unsaturated acids 3a-l were synthesized using published methods. 26 The 1 H-and 19 F-NMR spectra were recorded at 21 C on Bruker spectrometers operating at 400 MHz, 101 MHz and 600 MHz, 151 MHz, respectively. Enantiomer separations were obtained on Agilent 1260 HPLC instrument using either Chiralpak® ZWIX(+) (4 mm Â 250 mm) column and a mixture of methanol (containing 100 mmol L À1 formic acid and 50 mmol L À1 diethylamine), acetonitrile and water in proportion of 49 : 49 : 2 (v/v/v) as eluent at a ow rate of 1 mL min À1 or Crownpak® CR-I(+) column (150 Â 3.0 mm Â 5 mm) and a mixture of HClO 4 solution (3.6 g L À1 , pH 1.5): acetonitrile as mobile phase at a ow rate of 0.4 mL min À1 . NMR spectra and HPLC data are presented as ESI ‡ material.

Expression and purication of PaPAM
The gene of PAM from Pantoea agglomerans (encoding 541 AAs -Uniprot code: Q84FL5) was optimized to the codon usage of E. coli. The 1626 bps long synthetic gene was produced and cloned into pET-19b vector using the XhoI and Bpu1102I cloning sites. The recombinant PaPAM carrying an N-terminal (His) 10 -tag was produced in E. coli BL21(DE3)pLysS cells. For the expression step a colony of the transformed plasmid was grown overnight at 37 C in 5 mL of LB medium containing carbenicillin (50 mg mL À1 ) and chloramphenicol (30 mg mL À1 ). The overnight culture was added to LB medium (0.5 L) in an Erlenmeyer ask and grown at 37 C until OD 600 reached 0.7-0.8. Then the temperature was lowered to 25 C and the cells were induced by the addition of IPTG (1 mM). The culture was shaken at 220 rpm at 25 C for 19 h longer. All of the subsequent procedures were carried out in an ice-bath. The cells were harvested by centrifugation (25 min, 5000 Â g) and re-suspended in 50 mL of lysis buffer (150 mM NaCl, 50 mM TRIS, pH 7.5) supplemented with DNAse, RNAse, lysozyme, PMSF (2 mM) and an EDTA-free protease-inhibitor cocktail. The cells were then lysed by sonication and the cell debris was removed by centrifugation (10 000 Â g, 30 min).
The proteins were puried on a column lled with nickelnitrilotriacetic acid agarose gel (Ni-NTA) following the manufacturer's protocol. 27 The expressed protein was eluted from the column with imidazole (500 mM in low salt buffer, pH 7.5). The purity of the protein in the eluted fractions was veried by SDS-PAGE analysis. The protein fractions were dialyzed against phosphate-buffered saline (50 mM) at 4 C followed by concentration by centrifugal ultraltration (using verticallyoriented ultraltration membrane VIVASPIN 10 000 MWCO, 5000 Â g, 4 C, to nal concentration of 3-5 mg mL À1 ). The concentration of PaPAM in the nal solutions was determined by Bradford's method. 28 PaPAM-catalysed biotransformations of (AE)-a-and barylalanines Into the solution of the substrate (rac-1a-f, h, i, k, l and rac-2a-g, j, 4 mg) in (NH 4 ) 2 CO 3 buffer (100 mM, pH 8.0, 2 mL), PaPAM (1.6 mg) was added and the reaction mixture was stirred at room temperature for 20 h. For HPLC measurements reaction samples (30 mL) were taken and the enzyme was precipitated with 30 mL MeOH. Aer ltration, the samples were diluted with the corresponding mobile phase and injected on HPLC.
Prior to 1 H-and 19 F-NMR investigations the reaction was quenched with methanol, followed by ltration and evaporation of the solvent in vacuum. Deuterated sodium hydroxide (2% NaOD) solution was added and the spectrum (ESI ‡) was recorded at room temperature.
Biotransformation of (AE)-a-or b-(thiophen-2-yl)alanine (rac-1b or rac-2b) under various conditions pH change in the range of 7-9. Into the solution of the substrate (rac-1b or rac-2b, 4 mg) in (NH 4 ) 2 CO 3 buffer (pH 7.0, 8.0 or 9.0; 100 mM, 2 mL), PaPAM (1.6 mg) was added and the reaction mixture was stirred at room temperature for 20 h. The reaction was stopped by heating at 90 C for 10 min. The solvent was evaporated, the sample re-dissolved in NaOD solution (2%, 0.5 mL) and analysed by 1 H-NMR (data not shown).
Change of ammonium carbonate concentration in the range of 50-1000 mM. Into the solution of the substrate (rac-1b or rac-2b, 4 mg) in (NH 4 ) 2 CO 3 buffer (50, 100, 200, or 1000 mM; pH 8.0; 2 mL), PaPAM (1.6 mg) was added and the reaction mixture was stirred at room temperature for 20 h. The reaction was stopped by heating at 90 C for 10 min. Then the solvent was evaporated, the sample re-dissolved in NaOD solution (2%, 0.5 mL) and analysed by 1 H-NMR (see Table 3).

Molecular modelling of the covalent enzyme-substrate N-MIO complexes in PaPAM
The homotetrameric X-ray structure of PaPAM [PDB ID: 3UNV] 6 was completed and adjusted using the Protein Preparation Wizard 29 in four steps: (i) hydrogen atoms were added and bond orders were assigned, (ii) artefacts of the protein crystallization procedure were removed, except for two phosphate ions, (iii) 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 and (iv) a constrained minimization was performed. Protein pK a were predicted using PROPKA. 30 In the further modelling process for the N-MIO intermediates, in accordance with to the proposed mechanism (Fig. 1), Tyr78 was set deprotonated and Tyr320 protonated. Further details on the computational methods and the model will be published in a forthcoming paper.
The rened and completed X-ray 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. 31 The PaPAM model was solvated explicitly with water and additional ions were added with respect to the experimental assay conditions. The buffer solvated model was then equilibrated with a slightly modied default equilibration protocol, applying harmonic constraints to the Cartesian coordinates of protein heavy atoms. A spherical model of the active site with a radius of 27Å and centred 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 our substrates (S)-1a-l and (S)-2a-lwere created by our induced-t covalent docking protocol. This involved the creation of initial conformations of compounds (S)-1a-l and (S)-2a-l by docking with Glide program suite 32 into a modied and articially enlarged active site in which the residues Leu216, Ile219, Leu104, Val108, Met84, Leu421, Leu171, and Phe428 were exchanged to Ala residues, further the MIO prosthetic group was reduced to Thr + Gly and three water molecules in the active site were removed.
Aer having docked into the enlarged active site, all side chains and the MIO group were restored, a covalent bond between the nitrogen atom of the amino group and the exocyclic carbon of MIO was created, the covalently bound ligands and the residues in close proximity to them were minimized and nally, redundant conformations were eliminated with Macro-Model. 33 During restoring the mutated side-chains, conformations of several active site residues were predicted with Prime. 34 Aer replacing the three active site water molecules removed earlier, a nal minimization in the 6Å proximity of the covalently bound ligand using Prime 34 resulted in the nal models and energies. OPLS2005 force eld was applied in all molecular mechanics calculations and simulations.
Statistical tests (one-way ANOVA, Kruskal-Wallis ANOVA, median test and Mann-Whitney U tests) were carried out and Fig. 4 was created using Statistica. 35 Non-signicant Levene and Shapiro-Wilk tests justied the use of one-way ANOVA. The probability value of type I error (a) was chosen to be 0.05 in all the cases.