徐全乐等《Journal of Agricultural and Food Chemistry》2021年

论文题目：β‑Cyanoalanine Synthase Regulates the Accumulation of β‑ODAP via Interaction with Serine Acetyltransferase in Lathyrus sativus

论文作者：Yaoyao Song，Lei Wang，Fengjuan Liu，Chengjin Jiao，Hao Nan，Xiao Shen，Hong Chen，Yifan Li，Beilei Lei，Jinglong Jiang，Peng Chen and Quanle Xu

非蛋白氨基酸β-N-草酰-L-a,β-二氨基丙酸（β--N-oxalyl-L-a,b- diaminopropionic acid，β-ODAP）是山黧豆（ Lathyrus sativus ）中的重要神经激活性物质。长期、过量摄入山黧豆及β-ODAP会造成人畜运动神经元损伤性疾病，称为神经性山黧豆中毒。前期研究结果表明，β-ODAP的生物合成与硫代谢等基础代谢途径密切相关。本研究采用体外酶活检测证实了β-腈基丙氨酸合成酶（β-cyanoalanine synthase，β-CAS）是合成β-ODAP重要前体物质β-异噁唑-5-酮-丙氨酸（β-(isoxazoline-5-on-2-yl)-L-alanine，BIA）的关键酶。该酶表现为β-CAS和半胱氨酸合成酶（cysteine synthase，CS）两种酶活，属于磷酸吡哆醛（pyridoxal phosphate，PLP）依赖的双功能酶。多序列比对及酶活检测表明，M135、M235、S239是调控β-CAS:CS酶活性大小的关键氨基酸位点，β-CAS的M135T/M235S/S239T突变体主要表现为CS活性。利用Y2H、BiFC和pull-down证实，β-CAS与丝氨酸乙酰基转移酶（serine acetyltransferase，SAT）在线粒体（mitochondria）中形成半胱氨酸调控复合物mCRC（cysteine regulatory complex），并通过下调β-CAS、上调SAT酶活起到β-ODAP生物合成分子开关的作用。上述结果加深了对于山黧豆β-ODAP生物合成分子机制的理解。

论文摘要：β-N-Oxalyl-L-α,β-diaminopropionic acid (β-ODAP), found in Lathyrus sativus at first, causes a neurological disease,lathyrism, when over ingested in an unbalanced diet. Our previous research suggested that β-ODAP biosynthesis is related to sulfurmetabolism. In this study, β-cyanoalanine synthase (β-CAS) was confirmed to be responsible for β-ODAP biosynthesis via in vitroenzymatic analysis. LsCAS was found to be pyridoxal phosphate (PLP)-dependent via spectroscopic analysis and dual functional viaenzymatic activity analysis. Generation of a M135T/M235S/S239T triple mutant of LsCAS, which are the key sites to control theratio of CAS/cysteine synthase (CS) activity, switches reaction chemistry to that of a CS. LsCAS interactions were further screenedand verified via Y2H, BiFC and pull-down assay. It was suggested that LsSAT2 interacts and forms a cysteine regulatory complex (CRC) with LsCAS in mitochondria, which improves LsSAT while reduces LsCAS activities to affect β-ODAP content positively.These results provide new insights into the molecular regulation of β-ODAP content in L. sativus.

论文链接：https://dx.doi.org/10.1021/acs.jafc.0c07542