Copyright © Physiologia Plantarum 2000 PHYSIOLOGIA PLANTARUM 110: 164 – 171. 2000 Printed in Ireland —all rights resered ISSN 0031-9317 Degradation of lysine in rice seeds: Effect of calcium, ionic strength, S -adenosylmethionine and S -2-aminoethyl-L-cysteine on the lysine 2-oxoglutarate reductase-saccharopine dehydrogenase bifunctional enzyme S. A. Gaziola a , L. Sodek b , P. Arruda c , P. J. Lea d and R. A. Azevedo a, * a Depto de Gene ´tica, Escola Superior de Agricultura ‘Luiz de Queiroz’, Uni. de Sa ˜o Paulo, Piracicaba, S.P., CEP 13400 -970, Brazil b Depto de Fisiologia Vegetal, Uni. Estadual de Campinas, Campinas, S.P., CEP 13083 -970, Brazil c Depto de Gene ´tica e Eoluc ¸a ˜o, Uni. Estadual de Campinas, Campinas, S.P., CEP 13083 -970, Brazil d Department of Biological Sciences, Uniersity of Lancaster, Lancaster, LA14YQ, U.K. *Corresponding author, e -mail: raazeed@carpa.ciagri.usp.br Received 14 January 2000; revised 5 April 2000 Lysine biosynthesis has been extensively studied and the regu- was not. It would appear that the modulation by Ca 2 + and latory enzymes characterized in some of the most important ionic strength of LOR is a common feature among plant LOR enzymes. S -adenosylmethionine (SAM) did not produce any crop plants, however, much less is known about the lysine significant effect on either enzyme activity, indicating that it degradation pathway. Lysine 2-oxoglutarate reductase (LOR) only plays a role in the regulation of lysine biosynthesis. The and saccharopine dehydrogenase (SDH) have recently been effect of S -2-aminoethyl-L-cysteine (AEC) as both a substrate partially purified and characterized from plants, and have been shown to exist as a single bifunctional polypeptide. We have and an inhibitor of LOR activity was also tested. AEC was shown to partially substitute for lysine as a substrate for further characterized these enzymes from rice endosperm in relation to Ca 2 + and ionic strength modulation. Optimum pH LOR, but was also able to inhibit LOR activity, possibly values of 7.0 and 8.0 were obtained for LOR and SDH, competing with lysine at the active site. The higher K m for respectively. The LOR domain of the polypeptide was modu- AEC compared to lysine may reflect a lower binding affinity for AEC. lated by Ca 2 + and ionic strength, whereas the SDH domain tion has been shown to be considerably elevated in the majority of plant tissues analysed, all of them have failed to exhibit over-accumulation of lysine in the seed (Azevedo et al. 1990, Diedrick et al. 1990, Heremans and Jacobs 1995). It has been suggested that in order to achieve lysine accumu- lation in seeds, the catabolism of lysine must be reduced (Brochetto-Braga et al. 1992, Azevedo and Lea 2000). In the opaque -2 mutant of maize, the activities of two enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8; also des- ignated lysine  -ketoglutarate reductase [LKR]) and saccha- ropine dehydrogenase (SDH, EC 1.5.1.9), were shown to be strongly reduced, thus increasing the availability of lysine to be incorporated into storage proteins, or allowing more lysine to accumulate in the soluble form in the maize endosperm (Gaziola et al. 1999, Kemper et al. 1999). Falco Introduction Lysine is one of the essential amino acids synthesized via the aspartic acid metabolic pathway in plants (Azevedo et al. 1997) and is present in low concentration in cereal seeds (Azevedo and Lea 2000). In order to understand the factors governing lysine formation and accumulation, key enzymes responsible for the regulation of the aspartic acid metabolic pathway have been isolated and characterized in several plants species (Azevedo et al. 1997). However, most of the previous research has concentrated almost entirely on the enzymes controlling the biosynthesis of lysine, with special attention being paid to aspartate kinase (AK, EC 2.7.2.4) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52). Biochemical mutants containing enzymes of the aspartic metabolic pathway with altered regulatory properties have been selected in vitro, but although the threonine concentra- Abbreiations – AEC, S -2-aminoethyl-L-cysteine; LOR, lysine 2-oxoglutarate reductase; SAM, S -adenosylmethionine; SDH, saccharopine dehydrogenase. Physiol. Plant. 110, 2000 164