Department of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa, Alabama, USA.
Department of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa, Alabama, USA.
J Biol Chem. 2023 Mar;299(3):102966. doi: 10.1016/j.jbc.2023.102966. Epub 2023 Feb 1.
Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-dependent enzyme that uses cysteine to generate alanine and an active-site persulfide (C-S-S). The SufS persulfide is protected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "β-latch" structural element that includes the α helix, a glycine-rich loop, a β-hairpin, and a cis-proline residue. To identify a functional role for the β-latch, we used site-directed mutagenesis to obtain the N99D and N99A SufS variants. N99 is a conserved residue that connects the α helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a distorted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high-affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demonstrate that the β-latch does not affect the chemistry of persulfide formation but does protect it from undesired reductants. The data also indicate the β-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.
在氧化应激和铁饥饿条件下，大肠杆菌利用 Suf 途径组装铁硫簇。Suf 途径通过 SufS（一种 II 型半胱氨酸脱硫酶）动员硫。SufS 是一种依赖吡哆醛 5'-磷酸的酶，它使用半胱氨酸生成丙氨酸和活性位点过硫化物（C-S-S）。SufS 过硫化物受到外部氧化剂/还原剂的保护，需要转硫酶 SufE 接受过硫化物以完成 SufS 催化循环。最近关于 SufS 的报告确定了一个保守的“β-闩锁”结构元件，包括α螺旋、富含甘氨酸的环、β发夹和顺式脯氨酸残基。为了确定β-闩锁的功能作用，我们使用定点突变获得了 N99D 和 N99A SufS 变体。N99 是一个保守残基，通过氢键将α螺旋与富含甘氨酸的环的骨架连接起来。我们的 N99A 和 N99D SufS 的 X 射线晶体结构分别显示了扭曲的β发夹和富含甘氨酸的环，以及二聚体几何形状的变化。N99 变体的结构破坏允许外部还原剂 TCEP 与活性位点 C364-过硫化物中间体反应，在没有 SufE 的情况下完成 SufS 催化循环。这些取代似乎也破坏了高亲和力、紧密 SufS-SufE 复合物的形成，如荧光偏振所测量的。总的来说，这些发现表明β-闩锁不会影响过硫化物形成的化学性质，但会保护它免受不需要的还原剂的影响。数据还表明，β-闩锁在形成紧密 SufS-SufE 复合物以促进过硫化物转移方面发挥了意想不到的作用。
