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修饰基础代谢,而NFS1,一种人类CD,仅在[Acp]2:[ISD11]2:[NFS1]2复合物的形成中具有活性。尽管由于缺乏可获得的铁,在大肠杆菌细胞中积累了红色的IscS,正如我们之前的研究所揭示的,潜在的酶促反应机制尚不清楚.在这项研究中,IscS的N端与NFS1的C端融合,据报道,NFS1的C端几乎具有IscS的活性,并在395nm处显示出吡哆醛5'-磷酸(PLP)吸收峰。此外,SUMO-EH-IscS在iscS突变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,结果表明,IscSH104Q的新吸收峰,ISCSQ183E,IscSK206A,和IscSK206A和C328S变体在340和350nm可能对应于酶反应中间体,Cys-ketimine和Cys-aldimine,分别。然而,在保守的活性位点残基突变后,420和430nm处的其他吸收峰与活性位点口袋中的PLP迁移相关.此外,Cys-醌的相应吸收峰,Ala-ketimine,IscS中的Ala-醛亚胺中间体为510、325和345nm,分别,通过CD反应过程中的定点诱变和底物/产物结合分析确定。值得注意的是,通过在有氧条件下将IscS变体(Q183E和K206A)与过量的L-丙氨酸和硫化物一起孵育而在体外形成的红色IscS产生了与野生型IscS相似的吸收峰,在510nm。有趣的是,在Asp180和Gln183处具有与PLP的氢键的IscS的定点突变导致酶活性丧失,随后出现与NFS1一致的吸收峰(420nm)。此外,Asp180或Lys206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(His104,Asp180和Gln183)及其与IscSN末端PLP的氢键在确定L-半胱氨酸底物是否可以进入活性位点口袋和调节酶促反应过程中起关键作用。因此,我们的发现为评估保守活性位点残基的作用提供了一个框架,图案,和CD中的域。
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.