Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats: in vivo and in vitro studies.

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP) in the liver of adult rats increased 4-5 times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, in controlled by both glucagon and glucocorticoid.

Regulation of the lysine biosynthesis in Pichia guilliermondii.

The regulatory properties of four enzymes (homocitrate synthase, alpha-aminoadipate reductase, saccharopine reductase, saccharopine dehydrogenase) involved in the lysine biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory patterns found in other yeast species. The first enzyme of the pathway, homocitrate synthase, is feedback-inhibited by L-lysine. Some other amino acids (alpha-aminoadipate, glutamate, tryptophan, leucine) and lysine analogues are also inhibitors of one or more enzymes. It is shown that only the synthesis of homocitrate synthase is weakly repressed by L-lysine.

Lysine-ketoglutarate reductase and saccharopine dehydrogenase from Arabidopsis thaliana: nucleotide sequence and characterization.

We isolated the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, ED 1.5.1.9) from an Arabidopsis thaliana genomic DNA library based on the homology between the yeast biosynthetic genes encoding SDH (lysine-forming) or SDH (glutamate-forming) and Arabidopsis expressed sequence tags. A corresponding cDNA was isolated from total Arabidopsis RNA using RT-PCR and 5' and 3' Race. DNA sequencing revealed that the gene encodes a bifunctional protein with an amino domain homologous to SDH (lysine-forming), thus corresponding to LKR, and a carboxy domain homologous to SDH (glutamate-forming). Sequence comparison between the plant gene product and the yeast lysine-forming and glutamate-forming SDHs showed 25% and 37% sequence identity, respectively. No intracellular targeting sequence was found at the N-terminal or C-terminal of the protein. The gene is interrupted by 24 introns ranging in size from 68 to 352 bp and is present in Arabidopsis in a single copy. 5' sequence analysis revealed several conserved promoter sequence motifs, but did not reveal sequence homologies to either an Opaque 2 binding site or a Sph box. The 3'-flanking region does not contain a polyadenylation signal resembling the consensus sequence AATAAA. The plant SDH was expressed in Escherichia coli and exhibited similar biochemical characteristics to those reported for the purified enzyme from maize. This is the first report of the molecular cloning of a plant LKR-SDH genomic and cDNA sequence.

Synthesis of the Arabidopsis bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase enzyme of lysine catabolism is concertedly regulated by metabolic and stress-associated signals.

In plants, excess cellular lysine (Lys) is catabolized into glutamic acid and acetyl-coenzyme A; yet, it is still not clear whether this pathway has other functions in addition to balancing Lys levels. To address this issue, we examined the effects of stress-related hormones, abscisic acid (ABA), and jasmonate, as well as various metabolic signals on the production of the mRNA and polypeptide of the bifunctional Lys-ketoglutarate reductase (LKR)/saccharopine dehydrogenase (SDH) enzyme, which contains the first two linked enzymes of Lys catabolism. The level of LKR/SDH was strongly enhanced by ABA, jasmonate, and sugar starvation, whereas excess sugars and nitrogen starvation reduced its level; thus this pathway appears to fulfill multiple functions in stress-related and carbon/nitrogen metabolism. Treatments with combination of hormones and/or metabolites, as well as use of ABA mutants in conjunction with the tester sugars mannose and 3-O-methyl-glucose further supported the idea that the hormonal and metabolic signals apparently operate through different signal transduction cascades. The stimulation of LKR/SDH protein expression by ABA is regulated by a signal transduction cascade that contains the ABI1-1 and ABI2-1 protein phosphatases. By contrast, the stimulation of LKR/SDH protein expression by sugar starvation is regulated by the hexokinase-signaling cascade in a similar manner to the repression of many photosynthetic genes by sugars. These findings suggest a metabolic and mechanistic link between Lys catabolism and photosynthesis-related metabolism in the regulation of carbon/nitrogen partitioning.