Investigation On The Catalytic Function Of The BAHD3 Acyltransferase In β-ODAP Biosynthesis In Lathyrus Sativus L.

β-N-oxalyl-L-α,β-diaminopropionic acid（β-ODAP）,also known as dencichine,is endogenous active component of Lathyrus sativus L.（grass pea）,in addition,β-ODAP is found in rare Chinese herbal materials like as Panax notoginseng,Panax quinquefolium,panax ginseng and traditional crops like as Pisum sativum L.（pea）,andβ-ODAP has a potential function in the pathogenesis and treatment of neurodegenerative diseases.Radioisotope labeling indicated that L-2,3-diaminopropionic acid（L-DAP/L-DAPA）and oxalyl-Co A were direct precursors ofβ-ODAP biosynthesis.However,most researches aboutβ-ODAP biosynthesis bright to light serine acetyltransferase（SAT）andβ-cyanoalanine synthase（CASase）on the upstream of the metabolic pathway,the catalytic reactions of L-DAP and oxalyl-Co A are poorly studied.Recently,two task groups from UK and Israel infer BAHD acyltransferase may catalyzeβ-ODAP biosynthesis and ACYL ACTIVATING ENZYME3（AAE3）also as bifunctional enzyme catalyzeβ-ODAP biosynthesis.So which kind enzyme can catalyzeβ-ODAP biosynthesis?Here,we performed sequence alignment and phylogenetic tree analysis of BAHD acyltransferase of grass pea and pea and AAE gene family members,analyzed the docking ability of proteins（BAHD acyltransferase and AAE）to ligands（L-DAP and oxalyl-Co A）by protein sequence homology modeling and protein-ligand docking site prediction.Based on that,we illuminate function of BAHD acyltransferase and AAE by establishing the overexpression hairy root system of grass pea and pea,enzyme detection in vitro andβ-ODAP content analysis.Finally,we analyzed protein interaction network of BAHD acyltransferase based on IP-MS.The main results are as follows:（1）Based on BAHD acyltransferase with conserved motifs of HXXXD,DFGWG and YFGN,thirteen BAHDs sequences annotated in grass pea local transcriptome database,among them,the gene expression of Ls BAHD3 was higher during the seed development period.In like manner,we identified ten Ps BAHDs sequences from pea genome database.Sequence alignment and phylogenetic tree analysis indicates difference between Ls BAHD3 and Ps BAHDs.（2）Based on the amino acid sequence alignment of Ls BAHD3 and Ps BHADs,secondary structure analysis and 3D structure overlap analysis,it is speculated that the Ls BAHD3substrate binding pocket is specific and can specifically bind specifically to the substrates oxalyl-Co A and L-DAP.Molecular docking showed that the thiol parts of L-DAP and oxalyl-Co A were deeply buried in the Ls BAHD3 active cavity.Residues Asp166,Arg288 and Ala303deprotonate theβ-amino of L-DAP by forming a hydrogen bond with L-DAP.And the Ser372residue forms a hydrogen bond with the carboxyl terminus and thus activates oxalyl-Co A,promoting the occurrence of the catalytic reaction.（3）Oxalyl-Co A was Chemically synthesized using p-Thlocresol and oxalyl chloride as substrate and LC-MS/MS identified its mass-to-charge ratio（m/z）as 837.40,which is consistent with the theoretical value which is consistent with the theoretical value.On this basis,Ls BAHD3 and Ls AAE3 recombinant proteins were purified by prokaryotic expression and tested in vitro.At p H8.0,Ls AAE3 does not catalyzeβ-ODAP generation with sodium oxalate,Co A and L-DAP.Adding Ls BAHD3 to the reaction system,the generation ofβ-ODAP can be detected.This suggests that Ls AAE3 may catalyze the synthesis of oxalyl-Co A in an in vitro enzymatic reaction system,and then Ls BAHD3 enables the reaction of oxalyl-Co A with L-DAP to formβ-ODAP.（4）Construction of Ls BAHD3 protein HXXXD conserved motif targeted mutagenesis vector,that is p ET28a-Ls BAHD3-p.H162A,p ET28a-Ls BAHD3-p.D166A and p ET28a-Ls BAHD3-p.[H162A;D166A].And recombinant proteins were obtained using prokaryotic expression and tested for activity in vitro.At p H7.5,the wild Ls BAHD3 catalyzed the reaction of oxalyl-Co A with L-DAP to generateβ-ODAP,however,the catalytic activity of the above mutants was all significantly decreased.This indicates that the Ls BAHD3 conserved motif¹⁶²HSVVD¹⁶⁶ is important for the maintenance of protein catalytic function.（5）Hairy roots of grass pea and pea were obtained infected with Agrobacterium rhizogenes（C58C1）.Pea hairy root lines that were verified positive for genome,RNA and protein like as Ps BAHD3-16,Ps BAHD3-17,Ps BAHD3-24,Ps BAHD3-26,Ps BAHD3-27 were selected forβ-ODAP content determination.The results showed thatβ-ODAP synthesis could be detected in the above transgenic hairy root lines,whileβ-ODAP synthesis was not detected from pea line transformed by empty vector.Moreover,β-ODAP was significantly higher in overexpressed Ls BAHD3 positive lines like as OE Ls BAHD3-14 and OE Ls BAHD3-17 than control lines.This suggests that Ls BAHD3 hasβ-ODAP synthetase activity.（6）Positive lines of overexpression Ls BAHD3 transgenic lines was selected for IP-MS analysis and then obtained 110 proteins with potential interaction with Ls BAHD3.GO analysis indicates that proteins involved in nucleic acid binding,protein ubiquitination and phosphorylation maybe interact with Ls BAHD3.KEGG analysis shows that these proteins are mainly involved in energy metabolism,amino acid metabolism and protein transport and catalysis.Interestingly,the Ls AAE3 was screened in the IP-MS result.And then pull down result indicates an interaction between Ls AA3 and Ls BAHD3,indicating that Ls AAE3-Ls BAHD3 regulatesβ-ODAP generation as a complex.In conclusion,Ls AAE3 mainly plays oxalyl-Co A synthetase activity,and Ls BAHD3 may haveβ-ODAP synthetase activity;there is a protein interaction between Ls AAE3 and Ls BAHD3,which may work as a catalytic module to regulate the synthesis ofβ-ODAP.These results are beneficial to elucidating the biochemical mechanism ofβ-ODAP biosynthesis in grass pea.