Abstract
Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in animals and plants. To elucidate the biochemical signification of the linkage between the two enzymes of LKR/SDH, namely lysine ketoglutarate and saccharopine dehydrogenase, we employed various truncated and mutated Arabidopsis LKR/SDH polypeptides expressed in yeast. Activity analyses of the different recombinant polypeptides under conditions of varying NaCl levels implied that LKR, but not SDH activity, is regulated by functional interaction between the LKR and SDH domains, which is mediated by the structural conformation of the linker region connecting them. Because LKR activity of plant LKR/SDH enzymes is also regulated by casein kinase 2 phosphorylation, we searched for such potential regulatory phosphorylation sites using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and site-directed mutagenesis. This analysis identified Ser-458 as a candidate for this function. We also tested a hypothesis suggesting that an EF-hand-like sequence at the C-terminal part of the LKR domain functions in a calcium-dependent assembly of LKR/SDH into a homodimer. We found that this region is essential for LKR activity but that it does not control a calcium-dependent assembly of LKR/SDH. The relevance of our results to the in vivo function of LKR/SDH in lysine catabolism in plants is discussed. In addition, because the linker region between LKR and SDH exists only in plants but not in animal LKR/SDH enzymes, our results suggest that the regulatory properties of LKR/SDH and, hence, the regulation of lysine catabolism are different between plants and animals.
Highlights
Ductase (LKR)1 and saccharopine dehydrogenase (SDH) enzymes of the ␣-amino adipic acid pathway of lysine catabolism, a pathway that operates both in animals and plants [1, 2]
LKR Activity of the Arabidopsis Bifunctional Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) Is Modulated by Functional Interaction between the LKR and SDH Domains—To test whether the structural conformation of LKR/SDH is affected by interactions between different domains of this bifunctional protein, we have used yeast as an expression system to produce recombinant wild type and mutant forms of the Arabidopsis LKR/SDH enzyme, fused to a His tag
Because in vitro LKR activity of the maize LKR/SDH was previously shown to be stimulated by NaCl [13], we used different NaCl concentrations as a means to alter the structural conformation of the recombinant Arabidopsis LKR/SDH-derived proteins and tested their effects on LKR activity
Summary
Materials—Arabidopsis and maize plants were grown in a controlled greenhouse (16-h photo period at 25 Ϯ 5 °C). High Fidelity PCR system (Roche Diagnostics) was used for generating point mutation and constructs, The high molecular weight gel filtration calibration kit was purchased from Amersham Biosciences.
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