The β-latch structural element of the SufS cysteine desulfurase mediates active site accessibility and SufE transpersulfurase positioning.
The β-latch structural element of the SufS cysteine desulfurase mediates active site accessibility and SufE transpersulfurase positioning.
复制标题
DOI：
10.1016/j.jbc.2023.102966
复制
发表时间：
2023-03
影响因子：
4.8
通讯作者：
Frantom, Patrick A.
中科院分区：
文献类型：
--
作者：
Gogar, Rajleen K.;Carroll, Franki;Conte, Juliana, V;Nasef, Mohamed;Dunkle, Jack A.;Frantom, Patrick A.
在氧化应激和缺铁条件下，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）来动员硫。SufS是一种吡哆醛-5 ' -磷酸依赖酶，利用半胱氨酸生成丙氨酸和活性位点过硫化物（C364-S-S-）。SufS过硫化物不受外界氧化剂/还原剂的影响，需要过硫酶SufE接受过硫化物来完成SufS催化循环。最近关于suf的报道发现了一个保守的“β-闩锁”结构元件，包括α6螺旋、一个富含甘氨酸的环、一个β-发夹和一个顺式脯氨酸残基。为了确定β锁存器的功能作用，我们使用定点诱变获得了N99D和N99A SufS变体。N99是一个保守的残基，通过氢键将α6螺旋连接到富含甘氨酸的环的主链上。我们的N99A和N99D SufS的x射线晶体结构分别显示出扭曲的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 -过硫中间体反应，在没有SufE的情况下完成SufS催化循环。用荧光偏振测量，取代也似乎破坏了高亲和力，接近SufS-SufE复合物的形成。总的来说，这些发现表明β锁存器不影响过硫化物形成的化学性质，但确实保护过硫化物不受不需要的还原剂的影响。这些数据还表明，β锁闩在形成紧密的SufS-SufE复合物以促进过硫转移方面发挥了意想不到的作用。
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 (C364-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 α6 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 α6 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.
登录
查看更多内容
影响因子：
4.8
作者：
Kim, Sunmin;Park, SangYoun
通讯作者：
Park, SangYoun
影响因子：
5
作者：
Adinolfi S;Puglisi R;Crack JC;Iannuzzi C;Dal Piaz F;Konarev PV;Svergun DI;Martin S;Le Brun NE;Pastore A
通讯作者：
Pastore A
DOI：
10.1073/pnas.1909535116
发表时间：
2019-09-24
影响因子：
11.1
作者：
Patra, Shachin;Barondeau, David P.
通讯作者：
Barondeau, David P.
影响因子：
4.8
作者：
Blahut, Matthew;Wise, Courtney E.;Outten, F. Wayne
通讯作者：
Outten, F. Wayne
影响因子：
15
作者：
PETERSON, EA;SOBER, HA
通讯作者：
SOBER, HA