Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities

LsSAT2 (serine acetyltransferase in Lathyrus sativus) is the rate-limiting enzyme in biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP), a neuroactive metabolite distributed widely in several plant species including Panax notoginseng, Panax ginseng, and L. sativus. The enzymatic activity of LsSAT2 is post-translationally regulated by its involvement in the cysteine regulatory complex in mitochondria via interaction with β-CAS (β-cyanoalanine synthase). In this study, the binding sites of LsSAT2 with the substrate Ser were first determined as Glu²⁹⁰, Arg³¹⁶, and His³¹⁷ and the catalytic sites were determined as Asp²⁶⁷, Asp²⁸¹, and His²⁸² via site-directed/truncated mutagenesis, in vitro enzymatic activity assay, and functional complementation of the SAT-deficient Escherichia coli strain JM39. Furthermore, the C-terminal 10-residue peptide of LsSAT2 is confirmed to be critical to interact with LsCAS, and Ile³³⁶ in C10 peptide is the critical amino acid. These results will enhance our understanding of the regulation of LsSAT2 activities and the biosynthesis of β-ODAP in L. sativus.