Cloning of Chalcone–Flavanone Isomerase cDNA fromPueraria lobataand Its Overexpression inEscherichia coli

PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

8, 183–190 (1996)

0091

Cloning of Chalcone–Flavanone Isomerase cDNA from Pueraria lobata and Its Overexpression in Escherichia coli Yoshiya Terai, Isao Fujii, Soon-He Byun, Osamu Nakajima, Takashi Hakamatsuka,1 Yutaka Ebizuka,2 and Ushio Sankawa3 Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

Received February 12, 1996, and in revised form April 24, 1996

Chalcone–flavanone isomerase (CHI) cDNA was isolated from Pueraria lobata by combination of cDNA library screening using Phaseolus vulgaris CHI cDNA as a probe and polymerase chain reaction techniques. Analysis of nucleotide sequence of the cloned cDNA revealed a 675-bp open reading frame encoding a 225amino-acid polypeptide with a molecular weight of 23,803 Da. The CHI cDNA coding region was cloned into pET-3d expression vector and successfully overexpressed in Escherichia coli cells as active CHI enzyme. The recombinant CHI was then purified to apparent homogeneity by DEAE–cellulose column chromatography. Replacement of Cys-119 with Ala was carried out by site-directed mutagenesis and the result that the mutant CHI showed CHI enzyme activity confirmed that Cys-119 is not involved in the CHI catalytic active site. q 1996 Academic Press, Inc.

Flavonoids are important secondary metabolites widespread in the plant kingdom and exhibit versatile functions such as flower pigmentation, protection from UV, defense against pathogens, legume nodulation, and so on (1 – 3). Plants produce phytoalexins, low-molecular-weight antimicrobial compounds, when stressed by wounding or fungal infection. Production of isoflavonoid phytoalexin results from the coordinated induction of a series of biosynthetic enzymes (4–6). 1 Current address: Faculty of Pharmaceutical Sciences, Science University of Tokyo, Funagawara-cho 12, Shinjuku-ku, Tokyo 162, Japan. 2 To whom correspondence should be addressed. E-mail: yebiz@ mol.f.u-tokyo.ac.jp. 3 Current address: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 93001, Japan.

Chalcone–flavanone isomerase (CHI;4 EC 5.5.1.6) is an enzyme catalyzing the stereospecific isomerization of chalcones to their corresponding (2S)-flavanones (6). Coupled with chalcone synthase (CHS; EC 2.3.1.74), the first enzyme specific for flavonoid pathway, the second enzyme CHI has attracted much attention recently because of its involvement in the stress response in legumes (2). Induction of CHI activity, which precedes the accumulation of isoflavonoids, results from increased synthesis of the enzyme as a consequence of transient induction of mRNA encoding CHI (4,7). Mehdy and Lamb suggested that CHI may be a regulatory enzyme in the biosynthesis of isoflavonoid phytoalexins (8). CHI catalyzes the stereospecific formation of (2S)flavanone and increases its rate constant by 36 millionfold over that of the spontaneous reaction (9). However, CHI active site residue(s) and the mechanism responsible for this rate enhancement have not been clarified yet. Chemical modification studies using sulfhydryl selective reagents demonstrated that the cysteine residue located in or near the active site (10). It was also proposed that histidine residue was directly involved in the catalytic turnover by functioning as a nucleophile in the formation of a covalently bound intermediate based on the inactivation of CHI by diethyl pyrocarbonate which is reasonably selective reagent for histidine residue (11,12). However, Bednar and Adeniran reported that none of five histidine residues is essential for catalysis and suggested the general acid base catalysis mechanism (13). Bednar et al. also reported that chemical modification of cysteine residue did not abolish CHI catalytic activity (14). This result strongly indi4

Abbreviations used: CHI, chalcone–flavanone isomerase; CHS, chalcone synthase; MuMLV, Moloney murine leukemia virus: SDS, sodium dodecyl sulfate; PCR, polymerase chain reaction; IPTG, isopropyl-b-thiogalactopyranoside; LB, Lauria–Bertani medium.

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1046-5928/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIG. 1. Metabolic role of CHI in isoflavonoid biosynthesis in Pueraria lobata.

cated that the cysteine residue does not function as catalytic active site and the question of the nature of the CHI reaction mechanism is left open. Site-directed mutagenesis is a powerful tool for identifying essential active site residues. For this purpose, the gene should first be cloned and its expression system for active enzyme must be established. The genes coding for CHI have been cloned from several plant sources including Petunia hybrida and Phaseolus vulgaris (15–22). However, no successful expression of CHI enzyme has been reported. We have been studying isoflavonoid biosynthesis at the enzyme level in the cell suspension cultures of Leguminosae plant Pueraria lobata Ohwi, Japanese name ‘‘Kudzu,’’ a plant originally used in traditional oriental medicine, Puerariae radix (23–29) (Fig. 1). The main metabolites of the culture are daidzin and puerarin, 5-deoxy type isoflavone glycosides. In our previous enzymological studies, endogenous elicitor was used to induce CHS activity. Later, commercial yeast extract was found to be a more effective elicitor in inducing CHS activity. Thus, a cDNA library was constructed from the mRNA of yeast extract elicited P. lobata cell suspension cultures and a full-length CHS cDNA was cloned and sequenced (30,31). In this paper, we report the cloning and sequence analysis of CHI cDNA from P. lobata and its successful overexpression of the active CHI enzyme in Escherichia coli. Also, site-directed mutagenesis was carried out to

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confirm the result of Bednar et al. (14) that the cysteine residue is not involved in the CHI catalytic active site. MATERIALS AND METHODS

Enzymes and chemicals. Restriction endonucleases, AmpliTaq DNA polymerase, M13mp18 RF form, and pTV118N expression vector were purchased from Takara Shuzo (Japan). MuMLV reverse transcriptase was obtained from GIBCO-BRL. The [a-32P]dCTP and Hybond-N membrane were obtained from Amersham. Nytran membrane was from Schleicher and Schnell. pBluescripts and pET-3d were from Stratagene and Novagen, respectively. All other reagents were of molecular biology grade. Isolation of RNA and Northern blotting. Cell suspension culture of P. lobata was grown and elicited as previously described (25). Total RNA from yeast extract elicited cell suspension culture of P. lobata was extracted by phenol–SDS method (32). Northern blot analysis was carried out by the method described by Sambrook et al. (33). Screening of cDNA library. P. lobata cDNA library in lgt10 phage vector (30,31) was screened using a P. vulgaris CHI cDNA (8) as a probe. Labeling of the probe DNA with digoxigenin–dUTP was done with a DIG DNA labeling kit (Boehringer Mannheim). Hybridization was carried out in a solution containing 51 SSC (11 SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1%

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SDS, 0.1% blocking reagent (Boehringer Mannheim) solution, and 0.02% sodium N-lauroyl sarcosine at 607C overnight. Filters were washed twice with 0.11 SSC, 0.1% SDS at 607C for 30 min and then detected with a DIG nucleic acid detection kit (Boehringer Mannheim) using nitroblue tetrazolium salt and 5-bromo-4-chloro3-indolyl phosphate. Extension of 5* region and amplification by polymerase chain reaction. We synthesized two antisense primers, Rev-1, 5*-AAT CAT GGT CTC CAA CAC TG3 * and Rev-2, 5*-CTC TAT CAC TGC AGC CTC AT3 *, which correspond to nucleotide residues 645 – 626 and 606 – 587, respectively, of the CHI cDNA. Reverse transcription reaction mixture containing 68 mg of P. lobata total RNA, 1 pmol of Rev-1 primer, 4 ml of 250 mM Tris – HCl buffer, pH 8.6, containing 200 mM KCl, 5 mM MnCl2 , and 5 mM dithiothreitol, and 200 units of MuMLV reverse transcriptase in a volume of 20 ml was incubated at 377C for 1 h. The first strand cDNA was recovered by ethanol precipitation after phenol – chloroform extraction and dissolved in 11 ml of distilled water. Then, poly(dA) tails were added to the first strand cDNAs as follows. Tailing reaction mixture consisted of 11 ml of the first strand cDNA, 4 ml of 1 mM dATP, 4 ml of 1 M sodium cacodylate buffer, pH 7.2, containing 5 mM MgCl2 and 5 mM 2-mercaptoethanol, and 14 units of terminal deoxynucleotidyl transferase (GIBCO-BRL) and was incubated at 377C for 30 min and then heated at 657C for 5 min to inactivate the enzyme. After adding 80 ml of distilled water, 10 ml of the reaction mixture was used as template for the first PCR amplification with oligo(dT)15 and Rev-1 primers. PCR reaction mixture used was as follows: 10 ml of template cDNA solution, 100 pmol of each primer, 100 mM Tris – HCl buffer, pH 8.3, containing 500 mM KCl, 15 mM MgCl2 , and 0.01% gelatin, 8 ml of 2.5 mM dNTP, and 0.5 unit of AmpliTaq DNA polymerase in a total volume of 100 ml. Amplification was carried out at 947C for 40 s, at 557C for 1 min, and 727C for 2 min for 30 cycles. Then, second amplification was performed with 10 ml of the first PCR reaction mixture as template using Rev-2 and oligo(dT)15 primers. The amplified fragment of expected size (ca. 700 bp) was recovered from agarose gel and subcloned into pBluescript II vector. Sequences of insert cDNAs from several clones were determined to avoid Taq polymerase fidelity error. Construction of expression plasmids for CHI. Expression plasmid vectors used were pTV118N and pET3d. The chi Nco forward primer incorporated a NcoI restriction site (underlined) at the CHI cDNA ATG start codon (boldface): 5*-GA ATT CCC ATG GCG GCA GCA GCA GCA GTA G-3*. The chi Xba reverse primer incorporated a XbaI restriction site (underlined) and

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the complementary sequence corresponding to the stop codon TGA (boldface): 5*-GCA TGC TCT AGA TCA CTT TCC CTC AAC TCA-3*. The first strand cDNA reverse transcribed with oligo(dT)15 primer was amplified using the chi Nco forward and chi Xba reverse primers at 947C for 30 s, at 557C for 30 s, and at 727C for 1 min for 30 cycles. The reaction completed with a final 10min extension at 727C. The PCR amplified product was then subcloned into pBluescript II (pBS-CHI). The insert sequence was confirmed by DNA sequencing. The pBS-CHI insert was then cut off by NcoI and XbaI digestion and ligated into pTV118N digested with the same endonucleases to make pTV-CHI. Also, the pBS-CHI insert cut off by NcoI and BamHI digestion was ligated into pET-3d expression vector to obtain pET-CHI. Site-directed mutagenesis of CHI. To construct the expression plasmid for the mutant CHI of which cysteine-119 was replaced with alanine, site-directed mutagenesis was carried out as follows. Two primers were synthesized for this purpose. The C119A-Fsp antisense primer incorporated an FspI restriction site (underlined) 5* of the mutation site which alters the cysteine 119 (TGC) to a alanine (GCC) (box): 5*-GTG CGC AAC GGC GTT TTC CAT CAC CTT CTT3* (changed nucleotides in boldface). The C119A Fsp sense primer incorporated an FspI restriction site (underlined) 3* of the mutation site (Cys 119 to Ala; box): 5*-AAC GCC GTT GCG CAC ATG AAG TCT GTT GGG-3* (changed nucleotides in bold). The 5*-upper half of the CHI coding region from Cys119 was amplified from pBS-CHI with the chi Nco primer and the C119-Fsp antisense primer. The 3*lower half of the CHI coding region from Cys-119 was amplified with the chi Xba primer and C119A-Fsp sense primer. Each PCR product was then subcloned into pBluescript II to make pBS-C119A-FspF and pBSC119A-FspR. The insert of the pBS-C119A-FspF cut off by EcoRI and XbaI was ligated into EcoRI and XbaI digested M13mp18 to make M13-C119A-FspF. The insert of the pBS-C119A-FspR was cut off by FspI and ligated into the FspI site of M13-C119A-FspF. M13 recombinant with the reconstituted mutated CHI cDNA was selected and named M13-C119A. Presence of the designed mutation was confirmed by DNA sequencing. The C119A-CHI cDNA fragment was cut off by NcoI and BamHI digestion of the M13-C119A and ligated into pET-3d. Thus, the constructed plasmid for the C119A CHI expression was named pET-C119A. Expression and purification of the recombinant CHI. The seed culture of E. coli DH5a F* harboring the expression plasmid pTV CHI was grown at 377C for 15 h

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in LB broth with 100 mg/ml ampicillin and then inoculated into fresh LB broth with 100 mg/ml ampicillin. After 2 h incubation at 377C with shaking, IPTG was added to the culture (0.1 mM final concentration) to induce CHI protein expression. For the expression by pET-CHI, seed culture of E. coli BL21(DE3)pLysE harboring the pET-CHI was grown at 377C for 15 h in LB broth with 100 mg/ml ampicillin and 25 mg/ml kanamycin. LB broth (2 liters) containing 100 mg/ml ampicillin was inoculated with 12 ml of the seed culture and then grown for another 2 h at 377C. Then, IPTG was added to a final concentration of 0.4 mM. After incubation for 2 h, cells were harvested by centrifugation at 5000g for 5 min. Cells (6.4 g) were resuspended in 20 ml of 50 mM Tris–HCl buffer, pH 8.0, containing 1 mM EDTA and 100 mM NaCl. Then, lysozyme was added to a final concentration of 0.25 mg/ml. After being kept on ice for 20 min, sodium deoxycholate was added to a final concentration of 1 mg/ml and kept on ice for another 20 min. Then, the cell lysate was centrifuged to remove cell debris. To the resultant supernatant was added streptomycin to 20 mM. After removing the precipitated nucleic acids by centrifugation, the supernatant was subjected to ammonium sulfate precipitation. Precipitates with 80% saturation were dissolved in 6 ml of 25 mM potassium phosphate buffer, pH 7.5, and passed through PD-10 column equilibrated with the buffer. Eluate from the PD-10 column was then applied onto a DEAE–cellulose column (5 1 20 cm) equilibrated with 25 mM potassium phosphate buffer, pH 7.5. Elution was carried out with a linear gradient of KCl from 0 to 125 mM in the equilibration buffer (total 400 ml). CHI activity was eluted at around 80 mM KCl in a manner similar to that of the P. lobata CHI from cell suspension culture. DNA sequence analysis. Sequencing of DNA was carried out by dideoxy chain termination method using Sequenase Ver. 2.0 (Amersham) and fluorescein isothiocyanate-labeled primer or terminator (Yuki Gosei Kogyo Co. Ltd., Japan) with Hitachi Model SQ-3000 automated DNA sequencer. Fragments for sequencing were generated by restriction digestion. DNA sequence data were organized and analyzed using the DNASIS program (Hitachi Software Engineering Co. Ltd., Japan) and DNA analysis package of Human Genome Center, Institute of Medical Science, The University of Tokyo. Gel electrophoresis. SDS–PAGE analysis was carried out as described by Laemmli (34) and gels were stained with a silver stain kit of Kanto Chemicals (Japan) or Coomassie brilliant blue using Quick CBB kit from Wako Pure Chemicals Inc. (Japan). Assay of CHI activity. CHI activity was assayed spectrophotometrically by the modified method of Boland

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and Wong (35). To 2.91 ml of 0.1 M potassium phosphate buffer, pH 8.5, was added 50 ml of 12 mM isoliquiritigenin in ethyleneglycol monomethylether. An aliquot of enzyme (usually 40 ml) was added and the decrease of absorbance at 390 nm was monitored on spectrophotometer. All assays were carried out at 307C. RESULTS AND DISCUSSION

Isolation and Characterization of CHI cDNA clones A cDNA library was constructed using lgt10 phage vector from mRNA isolated from P. lobata cells 8 h after elicitation with yeast extracts. From this cDNA library, we cloned the P. lobata CHS cDNA (30,31). By screening of about 2 1 105 recombinant plaques of this P. lobata cDNA library, a single positive clone was obtained. Its insert cDNA was sequenced and found to contain 225-bp cDNA with a poly(A) tail. Homology with P. vulgaris CHI cDNA suggested that the cloned cDNA is a 3* end of the CHI cDNA of P. lobata. In order to obtain a full-length cDNA, amplification of the 5* end was carried out by rapid amplication of the cDNA ends method (36). Using two specifically designed antisense primers (Rev-1 and Rev-2) and oligo(dT)15 primer, a ca. 700-bp DNA fragment was amplified by PCR. Sequencing analysis of the PCR product showed that the cDNA was 659 bp long and overlapped over 50 bp with the previously cloned 225-bp CHI cDNA fragment. To avoid Taq DNA polymerase fidelity error, inserts of four independent clones were sequenced and consensus sequence was confirmed. By combination of these two cDNA fragments, the complete nucleotide sequence of the P. lobata CHI cDNA was delineated and submitted to nucleotide database with the Accession No. D63577. The P. lobata CHI cDNA has an overall length of 869 bp and the first ATG codon starts at the 55 base and ends with TGA stop codon at the 727 base. The sequence contains a 54-bp 5* noncoding region and a 140-bp 3* noncoding region with a part of a poly(A) tail (Fig. 2). The CHI cDNA open reading frame consisted of 675 bp which codes a 224-amino-acid polypeptide with a molecular weight of 23,803 Da. Comparison between P. lobata and P. vulgaris CHI cDNA open reading frame sequences showed high homology, 88.9% at the amino acid level and 86.9% at the nucleotide level, as expected from their close taxonomical relation, while about 50% homology was observed with P. hybrida CHI at the amino acid level. Expression of Recombinant CHI In order to construct a suitable expression system, we first tried expression with pTV118N expression vector (37). This vector contains a Plac promoter, a lacZ SD

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FIG. 2. Nucleotide and deduced amino acid sequence of CHI cDNA from Pueraria lobata. The nucleotides in the open reading frame are shown in capital letters. The positions of the possible translational initiation site and stop codon are underlined.

sequence, and a NcoI site at the lacZa start codon ATG. Introduction of NcoI site at the start codon ATG and XbaI site just after the stop codon TGA was accomplished by PCR. The amplified CHI cDNA NcoI–XbaI fragment was then introduced into the corresponding sites of pTV118N to construct the expression plasmid pTV-CHI (Fig. 3). E. coli DH5aF* transformant harboring pTV-CHI was subjected to IPTG induction at 0.1 mM. Analysis of the crude cell free extract showed the presence of CHI activity and a 24-kDa protein band on SDS–PAGE which was absent from extracts of control cultures of E. coli DH5aF* with or without pTV118N. CHI activity reached a maximum within 10 h after induction with IPTG. The expression of CHI enzyme activity proved that the cloned cDNA codes for CHI protein. However, the level of expression was not high enough for facile purification of the recombinant CHI. Therefore, we then tried to use pET expression vector for high level expression (38). A target gene cloned in the pET plasmid was expressed by strong T7 RNA polymerase in the host E. coli cell, usually BL21(DE3)

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which contains the polymerase gene under the control of the IPTG inducible lacUV5 promoter. The CHI expression plasmid pET-CHI was constructed by the introduction of the NcoI–BamHI CHI cDNA fragment of pBS-CHI into pET-3d corresponding cloning sites (Fig. 3). The pET-CHI was then used to transform the expression host E. coli BL21(DE3)pLysE. In order to induce CHI expression, IPTG (0.4 mM final concentration) was added to a culture of E. coli BL21(DE3)pLysE harboring pET-CHI. After 2 h induction, crude cell free extract was prepared and analyzed by SDS–PAGE. As shown in Fig. 4, a 24-kDa protein was expressed at very high level in soluble fraction with CHI enzyme activity. The recombinant CHI could be easily purified to apparent homogeneity by a simple two-step procedure of ammonium sulfate precipitation and DEAE–cellulose column chromatography. The fraction which showed the highest specific acitivity (3.67 kat/kg) contained 5.5 mg of apparently homogeneous recombinant CHI protein (Table 1). Its specific activity was comparable to that of highly purified P. lobata native CHI (3.73 kat/

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FIG. 3. Construction of CHI expression vectors. Pueraria lobata CHI cDNA coding region was amplified by PCR using restriction site designed primers (NcoI site in forward primer, XbaI site in reverse primer). After subcloning into pBluescript, CHI cDNA was cut off by NcoI and BamHI digestion and ligated into pET-3d to construct pET-CHI expression plasmid. Another expression plasmid pTV-CHI was constructed by NcoI and XbaI digestion and ligation into pTV118N.

kg) by an eight-step purification of 533-fold (unpublished result). Site-Directed Mutagenesis of CHI Several mechanisms have been proposed for the catalysis of CHI reaction. Hahlbrock et al. (39) pro-

posed a mechanism involving general acid base catalysis through an intermediate flav-3-en-4-ol. In contrast, Boland and Wong (11) suggested nucleophilic catalysis by an active site imidazole, followed by SN2 displacement by the 2 *-phenolate of the substrate. Alignment of the amino acid sequences deduced from CHI cDNA sequences so far cloned indicated the presence of consensus motif preserved around cysteine residue (Cys-119 of P. lobata CHI) as shown in Fig. 5. Dixon et al. concluded that CHI does not contain an essential cysteine residue based on the absence of enzyme inhibition in the presence of up to 0.1 M sodium tetrathionate (12,35,40). Also, Bednar et al. reported that the CHI derivatives with the chemically modified cysteine residue did not lose TABLE 1

Purification of the Recombinant Pueraria lobata CHI Expressed in Escherichia coli/pET CHI

FIG. 4. SDS–PAGE analysis of CHI expressed in E. coli transformants harboring pET-CHI by IPTG induction. M, molecular weight markers; C, total cell protein of the transformant before IPTG induction; T, total cell protein of the transformant 2 h after IPTG induction; P, purified recombinant CHI by DEAE–cellulose column chromatography.

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Purification

Total protein (mg)

Specific activity (kat/kg)

Crude extracta (NH4)2SO4 (0–80% sat.) DEAE–cellulose

270 231 5.5

1.11 1.09 3.69

a

Prepared from 6.4 g of E. coli cells.

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FIG. 5. Consensus amino acid sequence of CHI around conserved cysteine residue. Alignment of segments of the deduced amino acid sequences around the conserved cysteine residue (reverse face) in CHIs from Pueraria lobara (this paper), Antirrhinum majus (16), Malus sp. (17), Medicago sativa (18), Petunia hybrida (19,20), Phaseolus vulgaris (21), and Pisum sativum (22), is shown.

their catalytic activity (31). These results strongly indicated that the cysteine residue does not function as a catalytic active site. In order to conclude whether this cysteine is not actually involved in the catalytic reaction of CHI, site-directed mutagenesis was carried out. The mutant C119A CHI in which Cys-119 was replaced with alanine was prepared as described under Materials and Methods. The mutant C119A CHI was expressed in E. coli at the same high level and showed comparable activity (sp act, 0.97 kat/kg) to the normal CHI (1.1 kat/kg). Effects of SH inhibitors on CHI activity of both native and mutant recombinant enzymes were also examined. As shown in Table 2, the native CHI was sensitive to SH reagents and lost its activity almost completely as reported by Bednar et al. (10), but the mutant C119A CHI was found to be less sensitive. From these results, it is unambiguously concluded that Cys-119 is not catalytically involved in the CHI reaction as an active site residue but just locates near the active site. Further site-directed mutagenesis study is underway to identify active site residue to answer the mechanistic nature of the CHI reaction.

TABLE 2

Effect of SH Inhibitors on Pueraria lobata CHI Activity Inhibition (%) Inhibitor

Concentration (mM)

Wild-type CHI

C119A CHI

None HgCl2 Iodoacetamide N-ethylmaleimide

— 2.0 2.0 2.0

0 97 97 76

0 63 63 38

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ACKNOWLEDGMENTS We thank Professor Christopher J. Lamb of Salk Institute for Biological Studies (U.S.A.) for providing Phaseolus vulgaris CHI cDNA. We also thank the Human Genome Center, Institute of Medical Science, The University of Tokyo, for the use of nucleotide and protein sequence analysis program package.

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10. Bednar, R. A., Fried, W. B., Lock, Y. W., and Pramanik, B. (1989) Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site. J. Biol. Chem. 264, 14272–14276. 11. Boland, M. J., and Wong, E. (1979) Mechanism of action of chalcone isomerase. Bioorg. Chem. 8, 1–8. 12. Dixon, R. A., Dey, P. M., and Lamb, C. J. (1983) Phytoalexins: Enzymology and molecular biology. Adv. Enzymol. Relat. Areas Mol. Biol. 55, 1–136. 13. Bednar, R. A., and Adeniran, A. (1990) Chemical modification of chalcone isomerase by diethyl pyrocarbonate: Histidine residues are not essential for catalysis. Arch. Biochem. Biophys. 282, 393–398. 14. Bednar, R. A., McCafferey, and Shan, K. (1991) Introduction of unnatural amino acids into chalcone isomerase. Bioconjugate Chem. 2, 211–216. 15. Grotewold, E., and Peterson, T. (1994) Isolation and characterization of a maize gene encoding chalcone flavanone isomerase. Mol. Gen. Genet. 242, 1–8. 16. Podivinsky, E., Bradley, J. M., and Davis, K. M. (1993) Sequence announcements. Plant Mol. Biol. 21, 737–738. 17. McKhann, H. I., and Hirsh, A. M. (1994) Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): Highest transcript levels occur in young roots and root tips. Plant Mol. Biol. 24, 767–777. 18. van Tunen, A. J., Koes, R. E., Spelt, C. E., van der Krol, A. R., Stuitje, A. R., and Mol, J. N. M. (1988) Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: Coordinate, light-regulated and differential expression of flavonoid genes. EMBO J. 7, 1257–1263. 19. van Tunen, A. J., Hartman, S. A., Mur, L. A., and Mol, J. N. M. (1989) Regulation of chalcone flavanone isomerase (CHI) gene expression in Petunia hybrida: The use of alternative promoters in corolla anthers and pollen. Plant Mol. Biol. 12, 539–551. 20. Blyden, E. R., Doerner, P. W., Lamb, C. J., and Dixon, A. R. (1991) Sequence analysis of a chalcone isomerase cDNA of Phaseolus vulgaris L. Plant Mol. Biol. 16, 167–169. 21. Wood, A. J., and Davies, E. (1994) A cDNA encoding chalcone isomerase from aged pea epicotyls. Plant Physiol. 104, 1465– 1466. 22. Sparvoli, F., Martin, C., Scienza, A., Gavazzi, G., and Tonelli, C. (1994) Cloning and molecuar analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Mol. Biol. 24, 743–755. 23. Hakamatsuka, T., Noguchi, H., Ebizuka, Y., and Sankawa, U. (1988) Deoxychalcone synthase from cell suspension cultures of Pueraria lobata. Chem. Pharm. Bull. 36, 4225–4228. 24. Hakamatsuka, T., Noguchi, H., Ebizuka, Y., and Sankawa, U. (1989) Isoflavone synthase from cell suspension cultures of Pueraria lobata. Chem. Pharm. Bull. 37, 249–252. 25. Hashim, M. F., Hakamatsuka, T., Ebizuka, Y., and Sankawa, U. (1990) Reaction mechanism of oxidative rearrangement of flavanone in isoflavone biosynthesis. FEBS Lett. 271, 219–222.

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