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Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2023-05-10 , DOI: 10.1021/acs.jafc.3c00678 Hao Ma 1 , Yaoyao Song 1, 2 , Ying Zhang 1 , Huiying Guo 1 , Guowen Lv 1 , Hong Chen 1 , Jiayi Liu 1 , Xiaoning Liu 3 , Zhenfeng An 1 , Lei Wang 4 , Quanle Xu 1 , Chengjin Jiao 5 , Peng Chen 1
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2023-05-10 , DOI: 10.1021/acs.jafc.3c00678 Hao Ma 1 , Yaoyao Song 1, 2 , Ying Zhang 1 , Huiying Guo 1 , Guowen Lv 1 , Hong Chen 1 , Jiayi Liu 1 , Xiaoning Liu 3 , Zhenfeng An 1 , Lei Wang 4 , Quanle Xu 1 , Chengjin Jiao 5 , Peng Chen 1
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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.
中文翻译：
山黧豆丝氨酸乙酰转移酶关键位点及其酶活性的影响
LsSAT2（山黧豆中的丝氨酸乙酰转移酶）是 β- N-草酰-l - α,β-二氨基丙酸 (β-ODAP)生物合成的限速酶，β-ODAP 是一种神经活性代谢物，广泛分布于多种植物中，包括三七、人参，L. sativus。LsSAT2 的酶活性通过与 β-CAS（β-氰基丙氨酸合酶）的相互作用参与线粒体中的半胱氨酸调节复合物进行翻译后调节。在这项研究中，LsSAT2 与底物 Ser 的结合位点首先被确定为 Glu 290、 Arg 316和 His 317通过定点/截短诱变、体外酶活性测定和 SAT 缺陷大肠杆菌菌株 JM39 的功能互补，催化位点被确定为 Asp 267、Asp 281和 His 282。此外，LsSAT2 的 C 端 10 残基肽被证实对与 LsCAS 相互作用至关重要，而 C10 肽中的 Ile 336是关键氨基酸。这些结果将增强我们对 LsSAT2 活性调控和L. sativus中 β-ODAP 生物合成的理解。
更新日期：2023-05-10
中文翻译：
山黧豆丝氨酸乙酰转移酶关键位点及其酶活性的影响
LsSAT2（山黧豆中的丝氨酸乙酰转移酶）是 β- N-草酰-l - α,β-二氨基丙酸 (β-ODAP)生物合成的限速酶，β-ODAP 是一种神经活性代谢物，广泛分布于多种植物中，包括三七、人参，L. sativus。LsSAT2 的酶活性通过与 β-CAS（β-氰基丙氨酸合酶）的相互作用参与线粒体中的半胱氨酸调节复合物进行翻译后调节。在这项研究中，LsSAT2 与底物 Ser 的结合位点首先被确定为 Glu 290、 Arg 316和 His 317通过定点/截短诱变、体外酶活性测定和 SAT 缺陷大肠杆菌菌株 JM39 的功能互补，催化位点被确定为 Asp 267、Asp 281和 His 282。此外，LsSAT2 的 C 端 10 残基肽被证实对与 LsCAS 相互作用至关重要，而 C10 肽中的 Ile 336是关键氨基酸。这些结果将增强我们对 LsSAT2 活性调控和L. sativus中 β-ODAP 生物合成的理解。
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