Isolation of a New Dual-Functional Caffeine Synthase Gene Encoding
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FEBS 26861 FEBS Letters 534 (2003) 75^81 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Isolation of a new dual-functional ca¡eine synthase gene encodingprovided by Elsevieran - Publisher Connector enzyme for the conversion of 7-methylxanthine to ca¡eine from co¡ee (Co¡ea arabica L.)1 Kouichi Mizunoa;Ã, Akira Okudaa, Misako Katob, Naho Yoneyamab, Hiromi Tanakaa, Hiroshi Ashiharac, Tatsuhito Fujimuraa aInstitute of Agricultural and Forest Engineering, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan bGraduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan cDepartment of Biology, Faculty of Science, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan Received 10 September 2002; revised 6 November 2002; accepted 18 November 2002 First published online 11 December 2002 for example, Nazario and Lovatt proposed di¡erent pathways Abstract In co¡ee and tea plants, ca¡eine is synthesized from xanthosine via a pathway that includes three methylation steps. of ca¡eine biosynthesis in co¡ee leaves [5]. Since the ca¡eine We report the isolation of a bifunctional co¡ee ca¡eine synthase biosynthetic pathway contains three S-adenosyl-L-methionine (CCS1) clone from co¡ee endosperm by reverse transcription- (SAM)-dependent methylation steps, N-methyltransferases polymerase chain reaction (RT-PCR) and rapid ampli¢cation of play important roles. The available data indicate that a xan- cDNA ends (RACE) technique using previously reported se- thosineC 7-methylxanthosine C7-methylxanthineC theobro- quence information for theobromine synthases (CTSs). The pre- mineCca¡eine pathway is the major route to ca¡eine [3,4] dicted amino acid sequences of CCS1are more than 80% iden- with alternative entry to xanthosine 5P-monophosphate tical to CTSs and are about 40% similar to those of tea ca¡eine (XMP)C7-methyl XMPC7-methylxanthosine [6] (Fig. 1). synthase (TCS1). Interestingly, CCS1 has dual methylation ac- Recently we succeeded in highly purifying CS enzymes [7] tivity like tea TCS1. and in cloning a ca¡eine synthase (CS) gene from young tea ß 2002 Federation of European Biochemical Societies. Pub- lished by Elsevier Science B.V. All rights reserved. leaves [8]. We obtained homologous genes that encode caf- feine biosynthetic enzymes from young co¡ee leaves [9] and Key words: Co¡ee; Ca¡eine; N-Methyltransferase; independently Ogawa et al. also cloned similar genes using the 7-Methylxanthine; S-adenosyl-L-methionine; Theobromine sequence of our tea ca¡eine synthase (TCS) gene [10]. The latter genes encoded theobromine synthases named CTS1 and CTS2 [9] and CaMXMT by Ogawa et al. [10]. In contrast to tea TCS1 which catalyzes two ¢nal methylation steps of 1. Introduction ca¡eine biosynthesis, both from 7-methylxanthine to theobro- mine and from theobromine to ca¡eine, CTSs only catalyze Ca¡eine is a major and important e¡ective component of the conversion from 7-methylxanthine to theobromine and do co¡ee and is used widely in beverages. It is important to not have 1-N-methylation activity (Fig. 1). Since the co¡ee clarify the ca¡eine biosynthesis pathway and to clone the plant generally contains small amounts of theobromine and genes related to the production of ca¡eine not only to deter- accumulates high concentrations of ca¡eine, the co¡ee plant mine the metabolism of the purine alkaloid but also to control must have 1-N-methyltransferase that converts theobromine the content of ca¡eine in the co¡ee plant. to ca¡eine. After e¡orts to isolate 1-N-methyltransferase genes The metabolic pathway of purines, which family ca¡eine is from co¡ee, two new genes, tentatively named CtCS6 and a member of, has not been elucidated in detail, although we CtCS7, were isolated as homologs to CTS1 and CTS2. These recently proposed major biosynthetic pathways of the purine clones were successfully characterized as CS cDNAs from metabolites in leaves of co¡ee (Co¡ea arabica) and tea (Cam- young co¡ee beans and will be described here. ellia sinensis) [1^4]. Details of minor pathways for theobro- mine and ca¡eine are still obscure especially in co¡ee plants, 2. Materials and methods 2.1. Materials Co¡ee plants (C. arabica L.) were grown in a greenhouse at the *Corresponding author. Present address: Faculty of Bioresource Sci- Agricultural and Forest Research Center, University of Tsukuba, Ja- ences, Akita Prefectural University, Akita City, Akita 010-0195, Ja- pan. Immature seeds and young leaves were harvested and then stored pan. Fax: (81)-18-872 1677. at 380‡C until use. [Methyl-14C]SAM(55 mCi/mmol) was purchased E-mail address: [email protected] (K. Mizuno). from Amersham Pharmacia. 1 The nucleotide sequence data reported here will appear in the 2.2. Ampli¢cation of the coding sequences of co¡ee tentative ca¡eine EMBL, GenBank and DDBJ Nucleotide Sequence Databases under synthase (CtCS) cDNA using rapid ampli¢cation of cDNA ends the accession numbers AB086414 (CCS1) and AB086415 (CtCS7). (3P-RACE and 5P-RACE) Total cellular RNAs from immature co¡ee endosperm were ex- Abbreviations: CCS, co¡ee ca¡eine synthase; CtCS, co¡ee tentative tracted by cetyltrimethylammonium bromide (CTAB) solution ac- ca¡eine synthase; CTS, co¡ee theobromine synthase; RACE, rapid cording to the method of Chang et al. [11] with a slight modi¢cation. ampli¢cation of cDNA ends; SAM, S-adenosyl-L-methionine; TCS, First-strand cDNAs were synthesized using a 3P-RACE core set and tea ca¡eine synthase; XMP, xanthosine 5P-monophosphate oligo-dT 3 sites adaptor primer (oligo-dT 3SAP) (TaKaRa, Japan) as a 0014-5793 / 02 / $22.00 ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016S0014-5793(02)03781-X FEBS 26861 6-1-03 76 K. Mizuno et al./FEBS Letters 534 (2003) 75^81 2.3. Analysis of gene expression by semi-quantitative reverse transcriptase (RT)-PCR For semi-quantitative RT-PCR, total RNAs extracted from devel- oping endosperms, £ower buds and young leaves of co¡ee were treated with RNase-free DNase I (TaKaRa). DNA-free total RNA (382 ng) from each tissue was used for ¢rst-strand cDNA synthesis in 20 Wl reaction volume containing 2.5 units of AMV reverse transcrip- tase XL (TaKaRa) and 1 WMof oligo-dT 3SAP. The 20 Wl PCR reaction mixture contained 0.1 Wl of RT reaction mixture, 2.5 units of Gene Taq (Nippon Gene), 2.5 mMMgCl 2, 2 mMeach of deoxy- ribonucleotide triphosphate and 0.5 WMof gene-speci¢c primers. The gene-speci¢c primers used were 5P-GGCTTCTATGATAGTG-3P and 5P-CCTTTCCCCATATCTA-3P for CtCS6 and CtCS7; 5P-GGAG- GAAGGTTCTTTTG-3P and 5P-ATCGCCGTATACTTGGA-3P for CTS2. PCR run on a PTC-200 thermal cycler (MJ Research) using the following program: 94‡C for 1 min, 57‡C for 30 s, and 72‡C for 10 s. The ampli¢cation was done for 18^30 cycles and reaction tubes were removed every two cycles. The amplicons were 115 (CtCS6 and CtCS7) and 117 (CTS2) bp lengths, respectively. The ampli¢cation for each gene showed a linear curve. The reaction products were visual- ized on 10% polyacrylamide gels stained by ethidium bromide and ultraviolet (UV) transillumination. Semi-quantitative analysis of the intensify of £uorescence was conducted by a Macintosh computer using ‘NIH image’ software (http://rsb.info.nih.gov/nih-image/). 2.4. Construction of expression plasmids Expression plasmids for CtCS clones were constructed in a pET32a or pET23d vector (Novagen). To prepare expression plasmids for CtCS cDNAs, we carried out PCR-directed mutagenesis to produce an NcoI site at the translation initiation site in the cDNA sequence of CtCS clones. The primer carrying NcoI restriction sites (bold-faced type) for cloning were as follows: N1, 5P-CCATGGAGCTCCAA- GAAGTCC-3P. The pairs of primers were as follows: N1/CS10r for CtCS6 and CtCS7. The reaction program consisted of 30 cycles of 94‡C for 60 s, 55‡C for 60 s, and 72‡C for 90 s. The PCR products were subcloned into pGEM-T Easy vector (Promega). The subclones of the PCR products for CtCS6 and CtCS7 were digested with NcoI and SacI. The NcoI/SacI fragments were introduced into the pET32a or pET23d vector at the NcoI and SacI sites, respectively. The result- ing expression plasmids were introduced into the expression host Es- cherichia coli BL21(DE3). Fig. 1. Pathways for the biosynthesis of ca¡eine. Abbreviations: 2.5. Production of recombinant enzymes in E. coli and assay for enzyme GMP, guanosine 5P-monophosphate; XMP, xanthosine 5P-mono- activity phosphate; IMP, inosine 5P-monophosphate; AMP, adenosine 5P- A single colony of transformants was cultured at 37‡C overnight in monophosphate; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl- 3 ml of Luria broth containing 0.2 mg/ml ampicillin (LA) with con- C C L-homocysteine. A XMP 7-methyl XMP 7-methylxanthosine stant shaking. A portion (50 Wl) of the bacterial culture was added to pathway is operative in co¡ee leaves [6,12]. Ca¡eine synthase from 3 ml of fresh LA, and incubated at 37‡C for 2 h with shaking. Pro- tea leaves has broad substrate speci¢city and catalyzes the conver- duction of the recombinant proteins was induced by adding 9 Wlof sion of 7-methylxanthine to ca¡eine via theobromine (reactions I 0.1 Misopropyl- L-D-thio-galactopyranoside (¢nal concentration is 0.3 and II) [7]. mM), and cell growth was continued for 8 h at 25‡C. E. coli cells were harvested by centrifugation at 500Ug for 5 min, and then washed with 50 mMTris/HCl pH 8.5 containing 5 mMdithiothreitol primer.