Gene identification of the AAE superfamily and AAE3 activity determination in Lathyrus sativus L.

Abstract

[ Objective ] Dencichine ( β - N -oxalyl- L - α , β -diaminopropionic acid , β -ODAP ) is a neuroactive substance in species such as grass pea ( Lathyrus sativus L. ) and Panax notoginseng , and its biosynthesis is closely related to oxalyl-CoA. The aim of the study is to identify the acyl-CoA synthase encoding gene in grass pea based on the whole genome , which lays a foundation for the regulation of β -ODAP biosynthesis. [ Methods ] Acyl-activating enzyme superfamily genes were identified in the genome of grass pea using the AMP binding domain by hidden Markov model. The acyl-CoA synthase gene was cloned and analyzed via the multiple sequence alignment , prokaryotic expression , and enzyme activity detection. [ Results ] The re- sults showed that at least 22 AAE genes were located on 7 different branches in grass pea. Phylogenetic analysis indicated that LsAAE3 and Arabidopsis thaliana AtAAE3 clustered at one branch , belonging to acyl-CoA synthetase family. Cloning and sequence analysis showed that the full-length cDNA of LsAAE3 was 1 566 bp , and LsAAE3 and AtAAE3 possessed several conversed domains including AMP binding site , CoA binding site , and AAE consensus motif. The purified protein , which molecular weight was 56 kD , showed a single band as assayed by SDS-PAGE , which was detected in both induced bacterial proteins and purified recombinant proteins by Western blot , suggesting that the fusion protein was LsAAE3. The oxaloyl-CoA synthase activity of LsAAE3 depended on ATP and magnesium ions. [ Conclusion ] The study identified the acyl-activating enzyme ( AAE ) superfamily genes in the genome of grass pea and verified the oxaloyl-CoA synthase activity of LsAAE3 , paving the way for the analysis of LsAAE3 function in β -ODAP biosynthesis.

Keywords

• lathyrus sativus
• dencicine
• aae superfamily
• oxalyl-coa synthase
• enzyme activity detection