Lysine-Ketoglutarate Reductase Activity in Developing Maize Endosperm

Abstract

Lysine-ketoglutarate reductase activity was detected and characterzed in the developing endosperm of maize (Zea mays L). The enzyme showed specificity for its substrates: lysine, a-ketoglutarate, and NADPH. Formation of the reaction product saccharopine was demonstrated. The pH optimum of the enzyme was cdose to 7, and the Km for lysine and aketoglutarate were 5.2 and 1.8 millmolar, respectively. between 25 and 70%o saturation was collected and taken up in 2.5 ml of extraction buffer. After desalting on a Sephadex G-25 column (1.5 x 16 cm), the protein fraction was assayed for lysineketoglutarate reductase activity using conditions based on those described by Hutzler and Dancis (7). The assay mixture contained K-phosphate (pH 7.0; 300 ,umol), L-lysine (50 ,pmol), a-ketoglutarate (25 ,umol), NADPH (300 nmol), and enzyme (0.1-0.2 mg protein) in a total volume of 3 ml. The mixture was incubated at 30°C, and oxidation of NADPH was monitored in a spectrophotometer at 340 nm.Lysine-ketoglutarate reductase catalyzes the following reaction: lysine + a-ketoglutarate + NADPH -* saccharopine + NADP+ The enzyme has been characterized in human and animal tissues (3,7,8) where it is believed to catalyze the first step of lysine catabolism (4). A similar reaction involving NAD is catalyzed by saccharopine dehydrogenase, an enzyme found in yeast and fungi where the reverse of the above reaction is considered to be the final step of lysine biosynthesis (5,6,12,16). Thus, in some organisms saccharopine is an intermediate of lysine biosynthesis while in the others it appears to be involved in lysine breakdown. Actually, several pathways for lysine catabolism are known (11), but in higher plants no general pathway has been established. Nevertheless, tracer studies with barley seedlings have provided strong evidence for the pathway involving saccharopine as intermediate (10), suggesting that this route may be important for lysine breakdown in cereals at least. However, we are unaware of any studies with enzymes in higher plants in support of this pathway.In this report, we describe the characterization of lysine-ketoglutarate reductase in developing maize endosperm, a tissue known to degrade lysine extensively (14). MATERIALS AND METHODS RESULTS AND DISCUSSIONThe data in Table I show that partially purified extracts of maize endosperm oxidized NADPH in the presence of lysine and a-ketoglutarate. In the absence of one or both of these substrates, the oxidation of NADPH was minimal. Specificity for the substrates is indicated by the fact that several amino acids, including closely related structures such as L-ornithine and D-lysine, would not substitute for L-lysine, nor would oxaloacetate or pyruvate substitute for a-ketoglutarate. NADH would not replace NADPH as electron donor, as was found for the enzyme from human liver (7).To confirm that the activity being measured was truly lysineketoglutarate reductase, it would be essential to demonstrate the formation of saccharopine in the assay. This was attempted using I4CJlysine...

Partial Purification and Characterization of Lysine-Ketoglutarate Reductase in Normal and Opaque-2 Maize Endosperms

“…The maize enzyme exhibits specificity for lysine and a-ketoglutarate, NADPH is required as a cofactor, and saccharopine is the product of the reaction (2,3).…”

“…The first enzymatic evidence for the operation of the saccharopine pathway for lysine catabolism in plants was obtained with the demonstration of LKR3 activity in immature endosperm of maize (3). LKR (EC 1.5.1.8) condenses lysine and a-ketoglutarate into saccharopine using NADPH as cofactor.…”

Lysine degradation through the saccharopine pathway in mammals: involvement of both bifunctional and monofunctional lysine-degrading enzymes in mouse

“…This pathway has been described in plants [1][2][3][4] and mammals [5][6][7][8][9][10], and its first two reactions are catalysed by enzymic activities known as lysine-oxoglutarate reductase (LOR ; EC 1.5.1.8) and saccharopine dehydrogenase (SDH ; EC 1.5.1.9). The reductase activity condenses lysine and 2-oxoglutarate to form saccharopine [ε-N-(-glutaryl-2)--lysine].…”

“…Characterization of LOR activity in the immature maize endosperm [1,11] gave evidence for the operation of this lysine degradation pathway in plants. Both LOR and SDH activities reside on a single bifunctional polypeptide whose native form is a homodimer composed of identical 117 kDa subunits [4].…”

“…In baboon and bovine livers these two activities reside on a single polypeptide [14,15] and the bifunctional protein purified from these sources has been named aminoadipic semiAbbreviations used : LOR, lysine-oxoglutarate reductase ; PEG, poly(ethylene glycol) ; SDH, saccharopine dehydrogenase. 1 To whom correspondence should be addressed (e-mail parruda!turing.unicamp.br). The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers AF003551 (maize LOR/SDH and AJ224761) (cDNA-LOR/SDH, encoding the mouse LOR/SDH bifunctional enzyme).…”