Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues
Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues
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DOI：
10.1074/jbc.ra119.009471
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发表时间：
2019-08-16
影响因子：
4.8
通讯作者：
Outten, F. Wayne
中科院分区：
文献类型：
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作者：
Blahut, Matthew;Wise, Courtney E.;Outten, F. Wayne
铁硫（Fe-S）簇是许多金属蛋白正常运作所必需的。Fe-S簇（Isc）和硫利用因子（Suf）途径是大肠杆菌中产生这些Fe-S簇辅基的关键生物合成途径。虽然Isc在正常条件下占主导地位，但Suf在铁耗竭和氧化应激期间接管。通过这些系统的硫获取依赖于使用半胱氨酸脱硫酶机制从游离半胱氨酸中去除硫的能力。在Suf途径中，二聚体SufS蛋白使用辅因子吡哆醛5 '-磷酸（PLP）从游离半胱氨酸中提取硫，导致丙氨酸和过硫化物的产生。尽管取得了很大进展，但这种PLP依赖性酶的逐步机制仍不清楚。在这里，使用快速混合动力学结合X射线晶体学，我们分析了这个过程的预稳态动力学，同时分配早期中间体的机制。我们采用H123 A和C364 A SufS变体来捕获半胱氨酸脱硫酶反应的Cys-醛亚胺和Cys-酮亚胺中间体，从而能够直接观察这些中间体和SufS活性位点的相关构象变化。值得注意的是，我们提出，Cys-364是必不可少的定位Cys-醛亚胺为C α去质子化，His-123的作用，质子化的丙氨酸-烯胺中间体，和Arg-56促进催化通过氢键与巯基的Cys-醛亚胺。我们的研究结果，沿着与以前的SufS结构的发现，提出了一个详细的模型，SufS催化的反应，从Cys结合到C-S键裂解，并表明，Arg-56，His-123，和Cys-364是关键的SufS残基在这个C-S键裂解途径。
Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli. Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5 '-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steady-state kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for C alpha deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.