Roles of conserved active site residues in the IscS cysteine desulfurase reaction.
Roles of conserved active site residues in the IscS cysteine desulfurase reaction.
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IscS 半胱氨酸脱硫酶反应中保守活性位点残基的作用
DOI：
10.3389/fmicb.2023.1084205
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发表时间：
2023
影响因子：
5.2
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中科院分区：
文献类型：
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大肠杆菌半胱氨酸脱硫酶（CD），IscS，通过将硫（S）从L-半胱氨酸转移到许多细胞途径来改变基础代谢，而NFS 1，一种人类CD，仅在形成[Acp]2：[ISD 11]2：[NFS 1]2复合物中具有活性。尽管E.大肠杆菌细胞由于缺乏可获得的铁，正如我们以前的研究所揭示的，潜在的酶促反应的机制仍然不清楚。在这项研究中，IscS的N-末端与NFS 1的C-末端融合，据报道，NFS 1几乎完全具有IscS的活性，并在395 nm处显示吡哆醛5′-磷酸（PLP）吸收峰。此外，SUMO-EH-IscS在iscS突变体细胞中表现出显著的生长恢复和NADH-脱氢酶I活性。此外，通过体外和体内实验结合高效液相色谱和超高效液相色谱-串联质谱，表明IscS H104 Q、IscS Q183 E、IscS K206 A和IscS K206 A&C328 S变体在340和350 nm处的新吸收峰可能对应于酶反应中间体Cys-酮亚胺和Cys-醛亚胺，分别然而，保守的活性位点残基突变后，在420和430 nm处的额外吸收峰与活性位点口袋中的PLP迁移相关。此外，在CD反应过程中，通过定点诱变和底物/产物结合分析确定，IscS中Cys-喹喔啉、Ala-酮亚胺和Ala-醛亚胺中间体的相应吸收峰分别为510、325和345 nm。值得注意的是，通过在有氧条件下将IscS变体（Q183 E和K206 A）与过量的L-丙氨酸和硫化物一起孵育在体外形成的红色IscS在510 nm处产生与野生型IscS相似的吸收峰。有趣的是，IscS在Asp 180和Gln 183处与PLP形成氢键的定点突变导致酶活性丧失，随后出现与NFS 1一致的吸收峰（420 nm）。此外，Asp 180或Lys 206的突变抑制了IscS在体外与L-半胱氨酸（底物）和L-丙氨酸（产物）的反应。这些结果表明，保守的活性位点残基（His 104，Asp 180和Gln 183）和它们的氢键与PLP在N-末端的IscS发挥关键作用，在决定L-半胱氨酸底物是否可以进入活性位点口袋，并调节酶促反应过程。因此，我们的研究结果提供了一个框架，用于评估保守的活性位点残基，基序和结构域在CD中的作用。
Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5′-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography–tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.