Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum
Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum
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DOI：
10.1021/acs.biochem.8b00601
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发表时间：
2018-09-25
期刊：
影响因子：
2.9
通讯作者：
Hausinger, Robert P.
中科院分区：
文献类型：
--
作者：
Fellner, Matthias;Rankin, Joel A.;Hausinger, Robert P.
来自植物乳杆菌的LarE是一种ATP依赖的硫转移酶，在乳酸消旋酶的共价连接的镍钳核苷酸（NPN）辅助因子（吡啶3-硫酰胺-5-硫代羧酸单核苷酸）的生物合成过程中牺牲其Cys176硫原子形成脱氢丙氨酸（Dha）侧链。辅酶A （CoA）稳定LarE并与蛋白质形成CoA- cys176混合二硫化物。本研究展示了LarE/CoA复合物的晶体结构，揭示了蛋白质与CoA的相互作用，模拟了与ATP的结合。CoA弱抑制LarE活性，并且CoA过硫能够从Dha176形式的蛋白中部分再生功能性LarE。这种循环反应的生理相关性尚不清楚。发现了一种新的LarE形式，一种NPN-LarE共价加合物，解释了先前的结果，即乳酸消旋酶载蛋白的激活只需要分离的LarE。无活性的C176A变体的晶体结构显示出与野生型LarE基本相同的褶皱。创建了LarE的其他活性位点变体，并对其活性进行了表征，并根据结构分析了所有LarE变体。最后，将L. plantarum LarE结构与热厌氧细菌热糖olyticum LarE的同源性模型进行了比较，预测其活性位点含有3个半胱氨酸残基，并与其他具有相似折叠和多个活性位点半胱氨酸残基的蛋白质进行了比较。这些发现表明，一些LarE同源物可能不是牺牲的，而是通过使用过硫机制或在该位置上的[4Fe-4S]簇上的不稳定位点催化硫转移。
LarE from Lactobacillus plantarum is an ATP dependent sulfur transferase that sacrifices its Cys176 sulfur atom to form a dehydroalanine (Dha) side chain during biosynthesis of the covalently linked nickel-pincer nucleotide (NPN) cofactor (pyridinium 3-thioamide-5-thiocarboxylic acid mononucleotide) of lactate racemase. Coenzyme A (CoA) stabilizes LarE and forms a CoA-Cys176 mixed disulfide with the protein. This study presents the crystal structure of the LarE/CoA complex, revealing protein interactions with CoA that mimic those for binding ATP. CoA weakly inhibits LarE activity, and the persulfide of CoA is capable of partially regenerating functional LarE from the Dha176 form of the protein. The physiological relevance of this cycling reaction is unclear. A new form of LarE was discovered, an NPN-LarE covalent adduct, explaining prior results in which activation of the lactate racemase apoprotein required only the isolated LarE. The crystal structure of the inactive C176A variant revealed a fold essentially identical to that of wild-type LarE. Additional active site variants of LarE were created and their activities characterized, with all LarE variants analyzed in terms of the structure. Finally, the L. plantarum LarE structure was compared to a homology model of Thermoanaerobacterium thermosaccharolyticum LarE, predicted to contain three cysteine residues at the active site, and to other proteins with a similar fold and multiple active site cysteine residues. These findings suggest that some LarE orthologs may not be sacrificial but instead may catalyze sulfur transfer by using a persulfide mechanism or from a labile site on a [4Fe-4S] cluster at this position.