Identification and Characterization of an Oxalyl CoA-Synthetase from Grass Pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) is grown in many parts of the world as an agricultural crop for human and animal consumption. Although it is advantageous as a drought resistant legume crop that can withstand harsh environmental conditions, it also produces a neurotoxic compound, b-N-oxalyl-L-a,b-diaminopropionic acid (b-ODAP) that may cause a neurodegenerative syndrome, lathyrism, if consumed as a main diet component. The importance of developing a grass pea cultivar that is defective in the biosynthesis of b-ODAP led us to isolate and characterize a key enzyme implicated in its biosynthesis, oxalyl CoA-synthetase (OCS). The principal activity of OCS is to promote the catabolism of oxalate. Oxalate is a secondary metabolite involved in a number of physiological roles such as the regulation of calcium levels, the prevention of aluminum toxicity and plant defense from insects and herbivores. However, high levels of oxalate are toxic to cells and require tight regulation using catabolic enzymes such as OCS. We cloned the gene encoding OCS from L. sativus (LsOCS), expressed and purified it from E. colicells, and analyzed its kinetic efficiency with several di-carboxylic acids. We found its highest CoA-ligation activity with oxalate, but also promiscuous activities with other acids such as malonate and succinate. In addition, we crystalized the protein in its AMP-bound form and show that it had adopted a thioester-forming conformation. Our findings reveal a high degree of similarity in sequence, structure and catalytic efficiency between LsOCS and its homolog from A. thaliana.