Structure and regulation of the bifunctional enzyme lysine‐oxoglutarate reductase‐saccharopine dehydrogenase in maize

Abstract

The lysine-oxoglutarate reductase (LOR) domain of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase (LOR/SDH) from maize endosperm was shown to be activated by Ca 2ϩ , high salt concentration, organic solvents and Mg 2ϩ . The Ca 2ϩ -dependent enhancement of LOR activity was inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and calmidazolium. Limited proteolysis was used to assess the structure/function relationship of the enzyme. Digestion with elastase separated the bifunctional 125-kDa polypeptide into two polypeptides of 65 kDa and 57 kDa, containing the functional domains of LOR and SDH, respectively. Proteolysis did not affect SDH activity, while LOR showed a time-dependent and protease-concentration-dependent inactivation followed by reactivation. Prolonged digestion or increasing amounts of elastase produced a complex pattern of limit polypeptides derived from additional cleavage sites within the 65-kDa (LOR) and 57-kDa (SDH) domains. The SDH-containing polypeptides inhibited the enzymatic activity of LORcontaining polypeptides. When separated from the SDH domain by limited proteolysis and ion-exchange chromatography, the LOR domain retained its Ca 2ϩ activation property, but was no longer activated by high salt concentrations. These results suggest that the LOR activity of the native enzyme is normally inhibited such that after modulation, the enzyme undergoes a conformational alteration to expose the catalytic domain for substrate binding.

Manipulação de cereais para acúmulo de lisina em sementes

“…Recentemente também foi demonstrado em plantas que as atividades de LOR-SDH são afetadas diferentemente por Ca 2+ , força iônica e fosforilação (Karchi et al, 1995;Miron et al, 1997;Kemper et al, 1998;Gaziola et al, 2000). A atividade de LOR de milho mostrou ser modulada por Ca 2+ enquanto que a atividade de SDH não o foi (Kemper et al, 1998).…”

“…A atividade de LOR de milho mostrou ser modulada por Ca 2+ enquanto que a atividade de SDH não o foi (Kemper et al, 1998). O aumento de atividade de LOR dependente de Ca 2+ pode ser inibido por EGTA e antagonistas de calmodulina, contudo não foi observada a ligação de calmodulina no domínio LOR (Kemper et al, 1998).…”

“…Estes resultados foram também observados para a LOR-SDH de arroz . Também para força iônica foi observado que a atividade de LOR foi estimulada, enquanto que a atividade de SDH permaneceu inalterada, tanto em milho (Kemper et al, 1998) como em arroz . No caso das enzimas de tabaco e soja, a atividade de LOR mostrou ser modulada por fosforilação, mas não a atividade de SDH (Karchi et al, 1995;Miron et al, 1997).…”

Improved procedures for extraction of lysine 2‐oxoglutarate reductase/saccharopine dehydrogenase (LOR/SDH) enzyme fromPhaseolus vulgariscultivars

“…Lysine 2-oxoglutarate reductase (LOR) first combines lysine and 2-oxoglutarate into saccharopine, whereas sacharopine dehydrogenase (SDH) converts saccharopine into oc-aminoadipic semialdehyde and glutamate . LOR/SDH has been isolated from plant tissues which allowed the characterisation of some physical and biochemical properties, kinetics and modulation of LOR activity by calcium, ionic strength, and phosphorylation (Gonçalves-Butruille et al 1996;Kemper et al 1998;Gaziola et al 2000;Galili et al 2001a;Lugli et al 2002). Although LOR/SDH has been characterised in a few cereal crops (Azevedo & Lea 2001;Azevedo 2002), the purification and characterisation of LOR/SDH from a dicotyledonous plant species has only been carried out in detail in soybean and Arabidopsis thaliana (Galili et al 2001a).…”

Caracterização das enzimas chaves para o controle do metabolismo de lisina em milho (Zea mays L.) geneticamente modificado

“…Posteriormente, a sacaropina é hidrolisada à ácido α-aminoadípico e ácido glutâmico em uma reação catalisada pela enzima sacaropina desidrogenase (SDH, EC GAZIOLA et al, 1999;KEMPER et al, 1998).…”