Plant Molecular Biology 46: 749–760, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 749 β -Cyanoalanine synthase and cysteine synthase from potato: molecular cloning, biochemical characterization, and spatial and hormonal regulation Akiko Maruyama 1,3 , Kazuki Saito 1,∗ and Kimiharu Ishizawa 2 1 Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba Uni- versity, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan ( ∗ author for correspondence; e-mail ksaito@p.chiba- u.ac.jp); 2 Development Biology and Neuroscience, Graduate School of Life Science, Tohoku University, Sendai 980-8578, Japan; 3 present address: RIKEN Plant Science Center, 2-1, Hirosawa, Wako-shi, Saitama, 351-0198, Japan Received 20 November 2000; accepted in revised form 30 May 2001 Key words: β -cyanoalanine synthase, cysteine synthase, cyanide metabolism, ethylene, wounding, potato, Solanum tuberosum Abstract β -Cyanoalanine synthase (CAS, L-3-cyanoalanine synthase; EC 4.4.1.9) is the most important enzyme in cyanide metabolism. In addition to CAS, cysteine synthase (CS, EC 4.2.99.8) possesses CAS activity. To explore the physiological significance of cyanide metabolism, we isolated the cDNA clones corresponding to purified CAS (designated PCAS-1 and PCAS-2) and CS (designated PCS-1 and PCS-2) from potato using the information of these amino acid sequences. The recombinant proteins of PCS-1, PCS-2 and PCAS-1 catalyzed both CAS and CS reactions, although the ratios between CAS and CS activity were remarkably different. PCAS-1 preferred the substrates for the CAS reaction to the substrates for the CS reaction. From the kinetic characters and homology of amino acid sequences with known CS-like proteins, PCS-1, PCS-2 and PCAS-1 were identified as cytosolic CS, plastidic CS and mitochondrial CAS, respectively. The highest level of CAS activity, CAS protein and its mRNA were detected in potato buds. Stimulation of CAS activity and protein accumulation by ethylene without the concomitant increase of its mRNA suggested that ethylene induces CAS protein accumulation at the post- transcriptional level. Introduction β -Cyanoalanine synthase (CAS; EC 4.4.1.9) cat- alyzes the conversion of cyanide and cysteine to β - cyanoalanine and sulfide. CAS is widely distributed in higher plants and plays a crucial role in cyanide metabolism (Miller and Conn, 1980). Because of its exclusive localization in mitochondria, the main phys- iological role of CAS has been considered to be the detoxification of toxic cyanide produced in plant tis- sues (Hendrickson and Conn, 1969; Akopyan et al., The nucleotide sequence data reported will appear in the GenBank, EMBL and DDBJ Nucleotide Sequence Databases under the ac- cession numbers AB027000 (PCAS-1 cDNA), AB029338 (PCAS-2 cDNA), AB029511 (PCS-1 cDNA) and AB029512 (PCS-2 cDNA). 1975; Wurtele et al., 1985; Manning, 1988; Tittle et al. 1990). Cyanide is known to be produced from two main sources in higher plants, for example, the degra- dation of cyanogenic compounds (Poulton, 1990) and the oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) in ethylene biosynthesis (Yip and Yang, 1988). A large-scale degradation of cyanogenic com- pounds is usually prevented in intact plants by the sep- arate compartmentalization of cyanogenic compounds and the degradation enzymes on both tissue and sub- cellular levels (Poulton, 1990). In some cyanogenic plants such as Hevea and black cherry, cyanogenic compounds are accumulated in the seeds and hy- drolyzed through germination without cyanide ema-