Báo cáo khoa học: New application of firefly luciferase ) it can catalyze the enantioselective thioester formation of 2-arylpropanoic acid

New application of firefly luciferase ) it can catalyze the enantioselective thioester formation of 2-arylpropanoic acid Dai-Ichiro Kato1, Keisuke Teruya1, Hiromitsu Yoshida1, Masahiro Takeo1, Seiji Negoro1 and Hiromichi Ohta2 1 Graduate School of Engineering, University of Hyogo, Japan 2 Department of Biosciences and Informatics, Keio University, Japan Keywords acyl-CoA synthetase; enantiomer; firefly luciferase; kinetic resolution; nonsteroidal anti-inflamatory drugs Correspondence D.-I. Kato, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan Fax: +81 79 267 4891 Tel: +81 79 267 4969 E-mail: kato@eng.u-hyogo.ac.jp (Received 22 February 2007, revised 4 June 2007, accepted 5 June 2007) doi:10.1111/j.1742-4658.2007.05921.x We introduce a new application of firefly luciferase (EC 1.13.12.7). The firefly luciferases belong to a large superfamily that includes rat liver long-chain acyl-CoA synthetase (LACS1). LACS1 is the enzyme that is involved in the deracemization process of 2-arylpropanoic acid and catalyzes the enantioselective thioester formation of R-acids. Based on the similarity of the reaction mechanisms and the sequences between firefly luciferase and LACS1, we predicted that firefly luciferase also has thioesterification activ-ity toward 2-arylpropanoic acid. From an investigation using three kinds of luciferases from North American firefly and Japanese fireflies, we have confirmed that these luciferases exhibit an enantioselective thioester forma-tion activity and the R-form is transformed to a thioester in preference to the S-form in the presence of ATP, Mg2+, and CoASH. The enantiomeric excesses of unreacted recovered acid and thioester were determined by chi-ral phase HPLC analysis and the resulting 2-arylpropanoyl-CoAs were identified by high resolution mass spectroscopy. The Km and kcat values of thermostable luciferase from Luciola lateralis (LUC-H) toward ketoprofen were determined as 0.22 mm and 0.11 s)1, respectively. The affinity of keto-profen was almost the same of d-luciferin. In addition, the calculated E-value toward ketoprofen was approximately 20. These results suggest that LUC-H could catalyze the kinetic resolution of 2-arylpropanoic acid efficiently and would be a new option for the preparation of optically act-ive 2-substituted carboxylic acids. Firefly luciferase is a well-known enzyme that concerns in the bioluminescence reaction. It catalyzes the oxida-tion of firefly luciferin with molecular oxygen in the presence of ATP and Mg2+, resulting in luminescence [1–3]. The stereoselectivity of this bioluminescent reac-tion was investigated in detail by Seliger et al. [4], who found that the d-form is the specific substrate for the light emission reaction whereas the l-form is not used for the light-producing reaction. The bioluminescence reaction of firefly luciferase is composed of two reac-tion steps. The first step is the activation of the carb-oxyl group to form luciferyladenylate and the second is the light emission reaction via the oxidation of this intermediate. The substrate activation mechanism in the initial step is commonly observed in adenylate-forming enzymes, such as aminoacyl-tRNA synthetases, Abbreviations ACS, acyl-CoA synthetase; ee, enantiomeric excess; fenoprofen, 2-(3-phenoxyphenyl)propanoic acid; flurbiprofen, 2-(3-fluoro-4-phenyl-phenyl)propanoic acid; ibuprofen, 2-(4-isobutylphenyl)propanoic acid; ketoprofen, 2-(3-benzoylphenyl)propanoic acid; LACS1, long-chain acyl-CoA synthetase; LUC-H, thermostable luciferase from Luciola lateralis; naproxen, 2-(6-methoxy-2-naphthyl)propanoic acid; tropic acid, 2-hydroxymethylpropanoic acid. FEBS Journal 274 (2007) 3877–3885 ª 2007 The Authors Journal compilation ª 2007 FEBS 3877 New application of firefly luciferase D.-I. Kato et al. Fig. 1. Two-step mechanism of adenylate-forming enzymes, light emission reaction of firefly luciferase and thioesterification reac-tion of acyl-CoA synthetase. The initial step of these enzymatic reactions is the activa-tion of the carboxyl group to form acyl-adenylate intermediate. In the case of thio-esterification reaction, the acyl group is then transferred to the thiol group of CoASH to form a thioester (acyl-CoA), whereas light emission reaction was achieved by a multi-step oxidation with molecular oxygen of this intermediate followed by the production of oxyluciferin. acyl-CoA synthetases (ACSs), and nonribosomal pep-tide synthetases (Fig. 1) [5]. In comparison with the amino acid sequences of firefly luciferases and these enzymes, it has become apparent that luciferase shares the most significant similarities with ACSs. In addi-tion, the crystal structures of Photinus pyralis and Luciola cruciata luciferases, which were determined by Conti et al. [6] and Nakatsu et al. [7], respectively, were confirmed to have the same framework as that of epimerization, and hydrolysis of the thioester [14]. In these processes, LACS1 catalyzes the thioesterifica-tion reaction and this reaction is the only step that proceeds in an enantioselective manner [15,16]. Thus, the enantiomeric ratio in the reaction mixture gradu-ally shifts to one enantiomer with the progression of the reaction. The absolute configuration of compounds sometimes has a strong influence on the biological activity. For ACSs. These results indicate that firefly luciferase example, 2-arylpropanoic acid constitutes an important evolved from the same ancestral enzymes as ACSs and acquired a luminescent system in the course of evolu- group of physiologically active compounds, because both enantiomers display different activities in vivo. tion. The supporting evidence of this hypothesis was The S enantiomers are generally more important reported by Airth et al. [8]. They showed the bifunc-tionality of firefly luciferase (i.e. it could catalyze the thioester formation of dehydroluciferin as well as a bioluminescent ability). More recently, Oba et al. [9] found that firefly luciferase also has the ability to form thioesters of long-chain fatty acids. This report indica-ted that firefly luciferase also has an acyl-CoA synthe- because they are active as nonsteroidal anti-inflamma-tory drugs [17]. On the other hand, the R enantiomer of flurbiprofen has recently been paid much attention because of its anticancer activity as well as a potent reducer of beta amyloid, the main constituent of amy-loid plaques in Alzheimer’s disease [18]. Thus, the pre-paration of optically pure enantiomers is extremely tase activity toward an unnatural substrate. In important and many kinds of enzymatic approaches addition, Nakamura et al. [10] showed that luciferase exhibits bimodal action depending on the chirality of luciferin, in that it transforms l-luciferin into luciferyl-CoA whereas it oxidizes the d-form to oxyluciferin. In 1990, Suzuki et al. [11] reported that P. pyralis have been tried so far [19–21]. In the present study, we examined whether firefly luciferase has thioesterification activity that can enan-tiodifferentiate unnatural substrates, such as 2-aryl-propanoic acid. Based on the similarity of the reaction luciferase showed a significant sequence similarity mechanisms and the sequences between firefly lucif-with rat liver long-chain acyl-CoA synthetase erase and LACS1, we predicted that firefly luciferase (LACS1). LACS1 is the enzyme that is involved in the deracemization process of 2-arylpropanoic acid [12,13]. Deracemization is a reaction that inverts the chirality of either enantiomer of a racemate to the also has thioesterification activity toward 2-arylpropa-noic acid. From the investigation using three kinds of luciferases from the North American firefly and Japan-ese fireflies, we have confirmed that these luciferases other antipode, resulting in an optically active com- exhibit an enantioselective thioesterification activity pound starting from a racemic mixture. In the case of rat liver, the deracemization process is realized by three enzymatic reactions, such as thioesterification, and the R-form is transformed to a thioester in prefer-ence to the S-form in the presence of ATP, Mg2+, and CoASH. 3878 FEBS Journal 274 (2007) 3877–3885 ª 2007 The Authors Journal compilation ª 2007 FEBS D.-I. Kato et al. Results and Discussion Construction of a simple assay system To confirm whether luciferase could truly catalyze the New application of firefly luciferase starting material, the ee of the compounds in the two layers will increase. After the above mentioned separation procedure, the ee of the materials in the organic layer was measured. For this experiment, three kinds of luciferases, P. pylalis enantioselective thioesterification reaction of 2-aryl- luciferase, L. cruciata luciferase, and thermostable lucif- propanoic acid, racemic 2-phenylpropanoic acid, which has the simplest structure of 2-arylpropanoic acids, was selected as a substrate and the enantiomeric excess (ee) of the unreacted acid and thioester were measured using a chiral phase HPLC column (ee exhibits how excessive the amount of one enantiomer is compared to the other in an optically active compound; ee value erase from Luciola lateralis (LUC-H), were selected. LUC-H is a mutant enzyme of L. lateralis luciferase, with improved thermostability and resistance to a kind of surfactant as compared with the wild-type [22] and it has been used for hygiene monitoring and biomass assays based on the ATP-bioluminescence method [23,24]. In the presence of ATP, Mg2+, and CoASH, the can be determined by the equation: ee (%) ¼ optical purity of the recovered acid increased gradually |R ) S|⁄(R+S) · 100, where R and S are the respect-ive moles of enantiomers in a compound). The pro-duced thioester, however, could not be applied to the chiral phase HPLC column because of its polarity. Thus, the thioester and unreacted acid had to be separ-ated before the analysis. To separate these two com-pounds, a simple protocol was constructed. When the thioester was produced by the reaction, a large hydro- to the S-form (Table 1, entries 1–3). In particular, the ee value exceeded over 35% when the luciferases from Jap-anese fireflies, L. cruciata and LUC-H, were used. These results demonstrate that the luciferases of the Japanese fireflies can differentiate the chirality of 2-phenylpropa-noic acid, transforming the R-form to a thiol ester with CoASH. Photinus pyralis firefly, however, exhibits little enantioselectivity toward this substrate. This result is of philic group was introduced into the molecule as some interest because these three enzymes exhibit signifi- compared with a hydrophobic benzene ring of the sub-strate. When the reaction was stopped by hydrochloric cant amino acid sequence similarity with each other. As LUC-H exhibited the highest enantioselectivity toward acid, the carboxylate would be protonated to form a 2-phenylpropanoic acid, further investigations were free acid, which would be easily extracted into an organic layer. On the other hand, the thioester would remain in the aqueous phase because of the hydrophi-licity of its structure. Thus, the unreacted acid and the produced thioester can be separated by a simple extraction procedure. In addition, if luciferase has an mainly performed using this enzyme. LUC-H catalyzed kinetic resolution of 2-arylpropanoic acid To expand the applicability of the present reaction, ability to distinguish the absolute configuration of the other 2-arylpropanoic acids, such as flurbiprofen, Table 1. Enantioselectivity of firefly luciferase catalyzed thioesterification reaction of 2-arylpropanoic acid. The substrate was incubated at 30 °C for 60 min with LUC-H in the presence of ATP, CoASH and Mg2+. The ee of the recovered acid was determined by chiral phase HPLC analysis; the ee of the thioester was determined by chiral phase HPLC analysis after the hydrolysis of thioester linkage to form the corres-ponding acid. ND, not detected. Entry Substrate 1 2-phenyIpropanoic acid 2 2-phenyIpropanoic acid 3 2-phenyIpropanoic acid 4 Flurbiprofen 6 Iburprofen 6 Ketoprofen 7 Ketoprofen 8 Ketoprofen 9 Naproxen 10 2-phenyIbutanoic acid 11 Tropic acid 12 2-phenylpentanoic acid 13 2-phenyl-3-methylbutanoic acid 14 2-(4-chlorophenoxy)propanoic acid 15 2-methyl-3-phenylpropanoic acid Origin of luciferase Photinus pyralis Luciola cruciata LUC-H LUC-H LUC-H Photinus pyralis Luciola cruciata LUC-H LUC-H LUC-H LUC-H LUC-H LUC-H LUC-H LUC-H Recovered acid (% ee) 4.6 (S) 35.2 (S) 42.4 (S) 92.4 (S) 96.1 (S) 44.7 (S) 98.0 (S) 99.6 (S) 99.6 (S) 57.6 (S) 8.3 (R) Racemate Racemate Racemate Racemate Thioester (% ee) 6.2 (R) 32.2 (R) 67.3 (R) 51.9 (R) 55.1 (R) 32.5 (R) 52.0 (R) 44.6 (R) 69.5 (R) 36.0 (R) > 99.9 (S) ND ND ND ND FEBS Journal 274 (2007) 3877–3885 ª 2007 The Authors Journal compilation ª 2007 FEBS 3879 New application of firefly luciferase ibuprofen, ketoprofen, and naproxen, were selected and investigated. These acids constitute an important group of physiologically active compounds. Although the enantiomers of these compounds have been already prepared via lipase catalyzed kinetic resolution and recrystallization of diastereomeric mixtures, the yield and the ee of the products are not sufficiently high [25]. In case of LUC-H catalyzed thioester formation, D.-I. Kato et al. examined (Table 1, entries 10–15). The enantioselective thioester formation reactions also occurred when the a-methyl substituent of 2-phenylpropanoic acid was replaced with an ethyl or hydroxymethyl group. LUC-H could differentiate the chirality of 2-phenylbutanoic acid and the ee value of unreacted acid was almost the same as the case of 2-phenylpropanoic acid. In the case of 2-hydroxymethylpropanoic acid (tropic acid), however, its enantioselectivity toward these profen which has a hydroxyl group on the methyl group of compounds was significantly high and the ee of the recovered acids were over 90% ee (Table 1, entries 4, 5, 8 and 9). The absolute configuration of the recov-ered acids was also S, similar to the case of 2-phenyl-propanoic acid. Because the ee of the thioesters could not be deter-mined as they existed, the thioester bond was hydro- 2-phenylpropanoic acid, the thioester was recovered in enantiomerically pure form, whereas the reaction rate dramatically decreased. In both cases, enantioselectivi-ty was almost the same. Unfortunately, however, bul-kier substitutions could not be used by the enzyme; 2-phenylpentanoic acid and 2-phenyl-3-methylbutanoic acid were only recovered as racemates and the no lyzed and the ee of the resulting free acid was measured. product could be detected in the aqueous phase The two kinds of hydrolytic conditions, chemical (Table 1, entries 12 and 13). In addition, the reaction hydrolysis under high pH and enzymatic hydrolysis did not proceed by the introduction of a spacer under neutral pH, were examined, because there was some fear of racemization by the chemical hydrolysis. The pKa of the a-methine proton of the 2-arylpropa-noyl-CoA is approximately 10.3 [14] and the pH in the chemically hydrolyzed reaction mixture was >10. The produced acid was extracted by an organic solvent and its ee was determined by chiral phase HPLC analysis. Contrary to our expectations, the ee values were almost the same regardless of the conditions (data not shown). Based on these results, both hydrolytic methods are applicable to give free acid from the thioester. In addi-tion, the enatiomer ratios of these compounds were par-tial to the R-form. These results give support to the results of the HPLC analysis of unreacted acids, which indicate that the thioester formation reaction proceeds in the R-enantioselective manner (Fig. 2). Substrate specificity of LUC-H catalyzed thioester formation To investigate the substrate tolerance of LUC-H cata-lyzed thioester formation, the other substrates were Fig. 2. Luciferase catalyzed enantioselective thioesterification of 2-arylpropanoic acid. LUC-H exhibits an enantioselective thioesterifi-cation activity and the R-form is transformed to a thioester in pref- erence to the S-form in the presence of ATP, Mg2+, and CoASH. atom between the chiral center and the benzene ring (Table 1, entries 14 and 15). At present, we have no idea why LUC-H is not able to use these compounds. Besides how to distinguish the absolute configuration of substrates, these results were interesting from the view point of the molecular recognition of this enzyme. The 3D structural analysis may shed light on the answers of these questions. In the case of recombinant rat LACS1, the substrate specificity was not investigated in detail and only three kinds of substrate, ibuprofen, fenoprofen, and flurbi-profen, were examined [16]. With these substrates, flurbiprofen could not be used by LACS1. In the case of LUC-H, however, a series of 2-arylpropanoic acids, including flurbiprofen, were converted to the corres-ponding thioesters with good enantioselectivity and other derivatives were also converted. These results suggested that LUC-H has a greater potential for enantioselective thioester formation. Purification and identification of LUC-H produced 2-arylpropanoyl-CoA The solid-phase extraction method was used to purify the thioesters. Purified compounds were subjected to TLC analysis and the Rf values of each compound were in good agreement with the chemically synthes-ized authentic samples. These were also identified by the ESI-MS method based on orthogonal TOF-MS. The mass spectra of 2-arylpropanoyl-CoAs were dom-inated by ions that agreed with the calculated mass of singly charged ions of the corresponding thioester. The results are summarized in Table 2. The detected masses were within the limit of the accuracy of the instrument 3880 FEBS Journal 274 (2007) 3877–3885 ª 2007 The Authors Journal compilation ª 2007 FEBS D.-I. Kato et al. New application of firefly luciferase Table 2. Identification of 2-arylpropanoyl-CoA produced by LUC-H with TOF-MS analysis. For the detection of 2-arylpropanoyl-CoAs by TOF-MS analysis, see the Experimental procedures section. Entry Thioester 1 Flurbiprofenyl-CoA 2 Ibuprofenyl-CoA 3 Ketoprofenyl-CoA 4 Naproxenyl-CoA Predicted [M-H]– 992.1868 954.2275 1002.1910 978.1011 Observed [M-H]– 992.1832 954.2288 1002.1919 978.1935 Error (p.p.m.) )3.63 1.40 0.92 2.43 Table 3. Cofactor requirements analysis for ketoprofenyl-CoA for-mation by LUC-H. The substrate was incubated at 30 °C for 60 min with LUC-H. The ee of the recovered acid was determined by chiral phase HPLC analysis; the ee of the thioester was determined by chiral phase HPLC analysis after the hydrolysis of thioester linkage to form the corresponding acid. Relative activity was calculated on the basis of the ee of recovered acid. ND, not detected. Fig. 3. Kinetic study of ketoprofenyl-CoA synthetic activity on LUC-H. According to the Lineweaver–Burk plot, the Km, Vmax and Co-factor Entry ATP CoASH 1 + + 2 – + 3 + – 4 – – Recovered acid (% ee) 98.4 0.98 0.55 0.62 Thioester (% ee) 80.9 ND ND ND Relative activity (%) 100 1.0 0.6 0.6 kcat values for racemic ketoprofen were 0.22 mM, 110.3 nmolÆ min)1Æmg)1 protein, and 0.11 s)1, respectively. value was similar to that of d-luciferin for the bioluminescence reaction (Km ¼ 0.15 mm) (the data are reported in the product data sheet of LUC-H, which was provided by Kikkoman corporation), keto-profen and d-luciferin could be bound in the active site at the same magnitude of affinity. The Michaelis–Men- [26]. These results suggested that the purified com-pounds were definitely 2-arylpropanoyl-CoAs and that LUC-H is able to catalyze the enantioselevtive thio-esterification reaction of 2-arylpropanoic acid. Cofactor requirement experiments were also per-formed using ketoprofen as the substrate (Table 3). Based on the ee of the unreacted acids, a dramatical decrease was observed in the absence of ATP and⁄or CoASH. In addition, no peak of thioester product was detected in the aqueous layer. These results suggested that these cofactors were essential for the reaction and 2-arylpropanoyl-CoA was produced by way of an acyl-adenylate intermediate. Kinetic analysis of LUC-H catalyzed thioester formation The kinetic study of the formation of ketoprofenyl-CoA catalyzed by LUC-H was examined to obtain the detailed reaction information. The efficiency of thioest-er formation was calculated from the peak areas of the remaining acid and flurbiprofen as an internal stand-ard using reversed phase HPLC analysis. According to the Lineweaver–Burk plot, the Km and Vmax values for racemic ketoprofen were 0.22 mm and 110.3 nmolÆ min)1Æmg)1 protein, respectively (Fig. 3). The kcat value was calculated to be 0.11 s)1. Because the Km ten parameters of recombinant rat LACS1 for the thio-ester formation were reported by Sevoz et al. [16] and the values of Km and Vmax for (R)-ibuprofen and (R)-fenoprofen were 1.7 mm, 353 nmolÆmin)1Æmg)1 protein and 0.10 mm, 267 nmolÆmin)1Æmg)1 protein, respect- ively. From these data, it seems reasonable to assume that the specificity of LUC-H and rat LACS1 toward 2-arylpropanoic acid would be almost the same and hence LUC-H could catalyze the efficient thioester formation as well as recombinant rat LACS1. E-value calculation of LUC-H catalyzed thioester formation The stereoselectivity of recombinant rat LACS1 was very high and the complete separation of the absolute configuration of the substrates was carried out [16]. In the case of LUC-H, however, the enantioselectivity was not perfect and the S-form was also converted to a thioester (data not shown). To confirm the enantio-selective ratio of LUC-H catalyzed thioesterification of ketoprofen, the E-value was calculated. The E-value was used to evaluate the enantioselectivity of enzymat-ic kinetic resolution and was determined by using the ee and yield of the unreacted acid according to the method described by Chen et al. [27]. The calculated E-values in three conversion points are summarized in FEBS Journal 274 (2007) 3877–3885 ª 2007 The Authors Journal compilation ª 2007 FEBS 3881 ... - tailieumienphi.vn