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

enantioselective thioester formation of 2-arylpropanoic acid Dai-Ichiro Kato1, Keisuke Teruya1, Hiromitsu Yoshida1, Masahiro Takeo1, Seiji Negoro1 and Hiromichi Ohta2 1 Graduate School o

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

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 activareac-tion 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,

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 Kmand kcatvalues 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

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-isobutyl2-(3-fluoro-4-phenyl-phenyl)propanoic acid; ketoprofen, 2-(3-benzoyl2-(3-fluoro-4-phenyl-phenyl)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.

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

ACSs These results indicate that firefly luciferase

evolved from the same ancestral enzymes as ACSs and

acquired a luminescent system in the course of

evolu-tion The supporting evidence of this hypothesis was

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-tase activity toward an unnatural substrate In

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

luciferase showed a significant sequence similarity

with rat liver long-chain acyl-CoA synthetase

(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

other antipode, resulting in an optically active

com-pound starting from a racemic mixture In the case

of rat liver, the deracemization process is realized by

three enzymatic reactions, such as thioesterification,

epimerization, and hydrolysis of the thioester [14] In these processes, LACS1 catalyzes the thioesterifica-tion reacthioesterifica-tion and this reacthioesterifica-tion 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 example, 2-arylpropanoic acid constitutes an important group of physiologically active compounds, because both enantiomers display different activities in vivo The S enantiomers are generally more important 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 important and many kinds of enzymatic approaches 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 mechanisms and the sequences between firefly lucif-erase and LACS1, we predicted that firefly luciflucif-erase 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 thJapan-ese luciferases exhibit an enantioselective thioesterification activity and the R-form is transformed to a thioester in prefer-ence to the S-form in the presprefer-ence of ATP, Mg2+, and CoASH

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.

Results and Discussion

Construction of a simple assay system

To confirm whether luciferase could truly catalyze the

enantioselective thioesterification reaction of

2-aryl-propanoic acid, racemic 2-phenyl2-aryl-propanoic 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

can be determined by the equation: ee(%)¼

|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-philic group was introduced into the molecule as

compared with a hydrophobic benzene ring of the

sub-strate When the reaction was stopped by hydrochloric

acid, the carboxylate would be protonated to form a

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

ability to distinguish the absolute configuration of the

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 luciferase, L cruciata luciferase, and thermostable lucif-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 optical purity of the recovered acid increased gradually

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 some interest because these three enzymes exhibit signifi-cant amino acid sequence similarity with each other As LUC-H exhibited the highest enantioselectivity toward 2-phenylpropanoic acid, further investigations were mainly performed using this enzyme

LUC-H catalyzed kinetic resolution of 2-arylpropanoic acid

To expand the applicability of the present reaction, 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 Mg 2+ 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.

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,

however, its enantioselectivity toward these profen

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-lyzed and the ee of the resulting free acid was measured

The two kinds of hydrolytic conditions, chemical

hydrolysis under high pH and enzymatic hydrolysis

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

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), which has a hydroxyl group on the methyl group of 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 product could be detected in the aqueous phase (Table 1, entries 12 and 13) In addition, the reaction did not proceed by the introduction of a spacer 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

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, Mg 2+ , and CoASH.

[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 Kmand 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

value was similar to that of d-luciferin for the bioluminescence reaction (Km¼ 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-ten parameters of recombinant rat LACS1 for the thio-ester formation were reported by Sevoz et al [16] and the values of Kmand Vmax for 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 kinetenzymat-ic 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

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

Predicted [M-H] –

Observed [M-H] –

Error (p.p.m.)

1 Flurbiprofenyl-CoA 992.1868 992.1832 )3.63

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.

Entry

Co-factor

Recovered acid (% ee)

Thioester (% ee)

Relative activity (%)

Fig 3 Kinetic study of ketoprofenyl-CoA synthetic activity on LUC-H According to the Lineweaver–Burk plot, the K m , V max and

kcat values for racemic ketoprofen were 0.22 m M , 110.3 nmolÆ min)1Æmg)1protein, and 0.11 s)1, respectively.

Table 4 There are little changes for each conversion

point This result exhibits that the produced thioester

does not affect the enantioselectivity of the enzyme

and the R-acid would be converted to a thioester

approximately 20 times faster than the S-acid

pH profile of LUC-H catalyzed thioester formation

The activity-pH profile of the LUC-H catalyzed

thio-esterification reaction of ketoprofen over a wide range

is shown in Fig 4 The enzyme was highly active in the

alkaline pH region with the maximum activity at

pH 9–10 In the pH range over 10, the apparent

enzy-matic activities were reduced markedly This is because

of the spontaneous hydrolysis of the thioester linkage

In comparison with the bioluminescence reaction, the

enzyme kept its activity for thioester formation in a

wider range of pH and the optimal shifted to the basic

region

Thioesterification acitivity of L cruciata and

P pyralis luciferases From the above experiments (Table 1, entries 1–3), it was known that L cruciata and P pyralis luciferases also have an ability to catalyze the enantioselective thioesterification of 2-arylpropanoic acid, although with selectivity inferior to that of LUC-H To disclose whether these luciferases have an ability to catalyze the enantioselective thioester formation toward other compounds, these enzymes were analyzed Ketoprofen was used as a substrate Fortunately, those two luciferases exhibited the enantioselective thioester formation activity (Table 1, entries 6 and 7) The enantiopreference of these enzymes was the R-form, similarly to the case of LUC-H The reaction effi-ciency of L cruciata luciferase is the same level as LUC-H In addition, P pyralis luciferase could also differentiate between the enantiomers of the substrate The produced thioester was also isolated by the solid-phase extraction method and the mass value of the purified thioester was consistent with the calculated value of ketoprofenyl-CoA In the case of P pyralis luciferase, the E-value was calculated as 16 This result suggested that the enantioselectivity of P

pyral-is luciferase toward ketoprofen was the same as that of LUC-H, although the selectivity toward 2-phenylpropanoic acid was quite different from each other

Conclusions

We found a unique method for the preparation of optically active 2-arylpropanoic acid using a biolumin-escence enzyme Based on the similarity of the reaction mechanisms and the sequences between firefly lucif-erase and rat LACS1, we predicted that firefly luciflucif-erase would have an enzymatic activity of enantioselevtive thioester formation The three kinds of luciferases from the North American firefly and Japanese fireflies exhibit an enantioselective thioesterification activity toward 2-arylpropanoic acid and the R-form is trans-formed to a thioester in preference to the S-form in the presence of ATP, Mg2+, and CoASH The produc-tion of 2-arylpropanoyl-CoAs was confirmed by the high accuracy exact mass measurements by TOF-MS analysis Thermostable luciferase from L lateralis (LUC-H) exhibited thioesterification activity in high efficiency and the ee of unreacted recovered acid and thioester were relatively high The Km and kcat values toward racemic ketoprofen were determined as 0.22 mm and 0.11 s)1, respectively, and the E-value for this substrate was calculated to be 20 These results

Table 4 Determination of enantioselectivity (E-value) for ketoprofen

by LUC-H catalyzed thioester formation The substrate was

incuba-ted at 30 C for 60 min with LUC-H Conversion was calculated from

the yield of recovered acid Yield of the recovered acid was

determined by the reversed phase HPLC analysis in the presence

of flurbiprofen as an internal standard ee of the recovered acid was

determined by chiral phase HPLC analysis E-value was

calculated based on the conversion (c) and ee of the recovered

acid (ee(s)) according to the equation: E ¼ ln[(1 ) c)(1 ) ee(s))] ⁄

ln[(1 ) c)(1 + ee(s))] [27].

Entry

Conversion

(%)

Recovered acid (% yield)

ee

Fig 4 pH activity profile of LUC-H catalyzed thioester formation

(s) and bioluminescent reaction (d) The activity of thioester

forma-tion was determined by the comparison with the initial velocity of

the reaction.

suggested that LUC-H can be used for the kinetic

resolution of 2-arylpropanoic acid and will be the new

choice for the preparation of optically active

2-substi-tuted carboxylic acids

Experimental procedures

General methods

All chemicals were commercially available and used without

further purification unless otherwise noted Analytical TLC

was developed on E Merck Silica Gel 60 F256 plates

(0.25 mm thickness) Firefly luciferases from P pyralis

and recombinant luciferase from L cruciata were purchased

from Sigma (St Louis, MO, USA) and Wako (Osaka,

Japan), respectively Thermostable luciferase from

L lateralis(LUC-H) was thankfully gifted from Kikkoman

Corporation (Tokyo, Japan) HPLC analysis was carried

out with a Shimazu liquid chromatograph (LC-20AT) or

CO-8020 (Toso Co., Ltd, Tokyo, Japan) Mass spectra were

recorded on a Waters Flight Mass Spectrometer

LCT-Pre-mier (Waters Corp., Milford, MA, USA) Centrifugation

was carried out by high speed refrigerated microcentrifuges

(TOMY, Tokyo, Japan) with TMP-11 rotor Protein

con-centration was determined by the method of Bradford [28]

with the protein assay kit (Bio-Rad Laboratories, Inc.,

Hercules, CA, USA) and BSA was used as the standard

Authentic samples of 2-arylpropanoyl-CoA were chemically

synthesized according to the described method [29] The

tesB gene of Escherichia coli JM109 was cloned into the

NdeI and XhoI sites of pET23C(+) vector by the standard

procedure and the resulting plasmid, pTesB, was

trans-formed into E coli BL21(DE3) The induction of tesB

protein expression was performed by the addition of

0.5 mm IPTG at 25C in Luria–Bertani medium containing

50 mgÆmL)1of ampicillin

Luciferase catalyzed kinetic resolution of

2-arylpropanoic acid

To 100 mm potassium phosphate buffer (pH 7.0), racemic

2-arylpropanoic acid (0.25 mm), ATP (10 mm), CoASH

(2 mm), and MgCl2(10 mm) were added The final volume

of the reaction mixture was adjusted to 500 lL The

mix-ture was preincubated at 30C for 10 min The reaction

was started by the addition of the appropriate amount of

firefly luciferase (10–1000 lg protein) and incubated at

30C for 60 min After adding 100 lL of 2 m HCl and

1 mL of diethyl ether to the reaction, the mixture was

sha-ken and centrifuged at 17 610 g for 10 min The separated

organic layer was concentrated in vacuo and the ee was

an-alyzed by HPLC with a Daicel Chiral column (Daicel

Chemical Industries Ltd, Tokyo, Japan) using a mixture of

hexane⁄ 2-propanol ⁄ trifluoroacetic acid in an appropriate

proportion as the eluent

The ee of the thioester in the aqueous layer was deter-mined after hydrolysis of the thioester linkage Two kinds of hydrolytic conditions were examined: chemical hydrolysis, using 120 lL of 2 m NaOH incubation at room temperature for 30 min, and enzymatic hydrolysis, using residual aque-ous layer neutralized to pH 7 by addition of 2 m NaOH and

100 lL of cell free extract of tesB overexpressed E coli and incubation at 30C for 30 min After the hydrolysis, the reaction was quenched by adding 120 lL of 2 m HCl and

1000 lL of diethyl ether The mixture was shaken and cen-trifuged at 17 610 g for 10 min The separated organic layer was concentrated in vacuo and the ee was analyzed by HPLC with a Daicel Chiral column

The conditions and results of HPLC analysis were: for 2-phenylpropanoic acid, Daicel Chiralcel OD-H column (hexane⁄ 2-propanol ⁄ trifluoroacetic acid ¼ 100 ⁄ 1 ⁄ 0.1%, 1.0 mLÆmin)1, 210 nm), tR 26.5 min (R-form) and 32.5 min (S-form); for flurbiprofen, Daicel Chiralcel OD-H column (hexane⁄ 2-propanol ⁄ trifluoroacetic acid¼ 100 ⁄ 1 ⁄ 0.1%, 1.0 mLÆmin)1, 210 nm), tR31.9 min (R-form) and 37.1 min (S-form); for ibuprofen, Daicel Chiralcel OD-H column (hex-ane⁄ 2-propanol ⁄ trifluoroacetic acid ¼ 100 ⁄ 1 ⁄ 0.1%, 1.0 mLÆ min)1, 210 nm), tR 15.7 min (R-form) and 18.3 min (S-form); for ketoprofen, Daicel Chiralcel OJ-H column (hex-ane⁄ 2-propanol ⁄ trifluoroacetic acid ¼ 95 ⁄ 5 ⁄ 0.1%, 1.0 mLÆ min)1, 254 nm), tR 39.7 min (R-form) and 50.2 min (S-form); for naproxen: Daicel Chiralcel OD H column (hex-ane⁄ 2-propanol ⁄ trifluoroacetic acid ¼ 95 ⁄ 5 ⁄ 0.1%, 0.5 mLÆ min)1, 254 nm), tR 23.3 min (R-form) and 27.4 min (S-form); for 2-phenylbutanoic acid, Daicel Chiralcel OD-H column (hexane⁄ 2-propanol ⁄ trifluoroacetic acid ¼

100⁄ 1 ⁄ 0.1%, 1.0 mLÆmin)1, 210 nm), tR20.0 min (R-form) and 28.4 min (S-form); for tropic acid, Daicel Chiralcel OD-H column (hexane⁄ 2-propanol ⁄ trifluoroacetic acid ¼

95⁄ 5 ⁄ 0.1%, 1.0 mLÆmin)1, 254 nm), tR 20.9 min (S-form) and 25.9 min (R-form); for 2-phenylpentanoic acid: Daicel Chiralcel OD-H column (hexane⁄ 2-propanol ⁄ trifluoroacetic acid¼ 100 ⁄ 1 ⁄ 0.1%, 1.0 mLÆmin)1, 210 nm), tR 17.9 min (R-form) and 23.6 min (S-form); for 2-phenyl-3-methylbuta-noic acid: Daicel Chiralcel OD-H column (hexane⁄ 2-propanol⁄ trifluoroacetic acid ¼ 100 ⁄ 1 ⁄ 0.1%, 1.0 mLÆmin)1,

210 nm), tR 16.3 min (R-form) and 25.0 min (S-form); for 2-(4-chlorophanoxy)propanoic acid: Daicel Chiralcel OJ-H column (hexane⁄ 2-propanol ⁄ trifluoroacetic acid¼ 95 ⁄ 5 ⁄ 0.1%, 1.0 mLÆmin)1, 254 nm), tR 15.4 min (R-form) and 19.0 min (S-form); for 2-methyl-3-phenylpropanoic acid, Daicel Chiralcel OJ-H column (hexane⁄ 2-propanol ⁄ tri-fluoroacetic acid¼ 95 ⁄ 5 ⁄ 0.1%, 0.5 mLÆmin)1, 254 nm), tR 20.0 min (R-form) and 22.4 min (S-form)

Purification and identification of 2-arylpropanoyl-CoA

2-Arylpropanoyl-CoA thioester was purified by solid-phase extraction cartridges (Chromabond C 18 ec,

Macherey-Nagel, Du¨ren, Germany) [30] After the

prepar-ation of the aqueous layer as described above, reaction

mix-ture was neutralized by the addition of 2 m NaOH and

ammonium acetate was added to a final concentration of

520 mm After cartridge preconditioning with consecutive

washing with methanol, water, and 520 mm ammonium

acetate solution (3 mL each), the mixture was loaded onto

the cartridge The column was rinsed with 5 mL of 520 mm

ammonium acetate solution to remove free CoASH The

thioester was recovered by elution with 5 mL of distilled

water The eluates were lyophilized and resolved in a small

amount of water Fractions containing the thioester, which

were identified with delayed nitroprusside reaction [31],

were checked by TLC and TOF-MS analysis TLC was

performed on silica gel plates using the solvent system of

1-butanol⁄ water ⁄ acetic acid (60 ⁄ 35 ⁄ 25) and Rf values of

0.57, 0.56, 0.55, and 0.56 were observed for

flurbiprofenyl-CoA, ibuprofenyl-flurbiprofenyl-CoA, keoprofenyl-flurbiprofenyl-CoA, and

naproxe-noyl-CoA, respectively Purified thioesters were subjected to

ESI-TOF-MS with an LCT-Premier (Waters Corp.) The

data were acquired in the negative ESI mode and leucine

enkephalin was selected as a reference compound for high

accuracy exact mass measurements

Kinetic analysis

To 100 mm potassium phosphate buffer (pH 7.0), racemic

ketoprofen (0.04–1.0 mm), ATP (10 mm), CoASH (2 mm),

and MgCl2 (10 mm) were added The final volume of the

reaction mixture was adjusted to 500 lL The mixture was

preincubated at 30C for 10 min The reaction was started

by the addition of LUC-H (28.5 lg protein) and incubated

at 30C for 10 min After adding 100 lL of 2 m HCl and

1000 lL of diethyl ether to the reaction, the mixture was

shaken and centrifuged at 17 610 g for 10 min The

separ-ated organic layer was concentrsepar-ated in vacuo The yield of

the unreacted carboxylic acid was measured by

reversed-phase HPLC using a Mightysil RP-18GP Aqua 250–4.6

(5 lm) (Kanto Corp., Tokyo, Japan) with an isocratic

solvent system of water⁄ acetonitrile ⁄ trifluoroacetic acid

(2⁄ 3 ⁄ 0.05%) at a flow rate of 0.5 mLÆmin)1 at 35C The

fractions from the column were monitored at 254 nm and

flurbiprofen was used as an internal standard Retention

times of ketoprofen and flurbiprofen were 9.5 min and

13.2 min, respectively Each assay was performed eight

times The kinetic parameters for racemic ketoprofen were

determined by the method of Lineweaver–Burk plots

E-value calculations

The reaction condition was the same as the kinetic analysis,

except that 140 lg protein of LUC-H was used for this

study Each assay was performed three times The E-value

was calculated from the ee and yield of the unreacted acid

according to a previously described method [27]

pH profile experiments

The thioester formation activity was determined in the following buffers (100 mm): citric acid–sodium citrate buffer (pH 3.0–4.0), succinate–KOH buffer (pH 4.0–5.5), Me–KOH buffer (pH 5.5–7.0), potassium phosphate buffer (pH 7.0–8.0), Tris⁄ HCl buffer (pH 8.0–10.0), sodium car-bonate–sodium hydrogen carbonate buffer (pH 10.0–11.5) The reaction condition was the same as the kinetic analysis, except that other buffer system and 280 lg protein was used for this experiment Each assay was performed three times and the initial velocity of the thioester formation was determined at each pH Relative activity was calculated from the comparison of these velocities

Acknowledgements

This work was partially received the financial support from a Kawanishi Memorial Shinmaywa Education Foundation DK greatly acknowledges the helpful sup-port of Mr Mikio Bakke of Kikkoman Corporation

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