Structure and Substrate Specificity of the Pyrococcal Coenzyme A Disulfide Reductases/Polysulfide Reductases (CoADR/Psr): Implications for S0-Based Respiration and a Sulfur-Dependent Antioxidant System in Pyrococcus
Structure and Substrate Specificity of the Pyrococcal Coenzyme A Disulfide Reductases/Polysulfide Reductases (CoADR/Psr): Implications for S0-Based Respiration and a Sulfur-Dependent Antioxidant System in Pyrococcus
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DOI：
10.1021/bi3014399
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发表时间：
2013-04-23
期刊：
影响因子：
2.9
通讯作者：
Crane, Edward J., III
中科院分区：
文献类型：
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作者：
Herwald, Sanna;Liu, Albert Y.;Crane, Edward J., III
FAD和NAD(P)H依赖的辅酶A二硫化物还原酶/多硫化物还原酶(CoADR/PSR)被认为在硫磺还原过程中起重要作用，而在厌氧嗜热杆菌中，FAD和NAD(P)H依赖的辅酶A二硫化物还原酶(CoADR/PSR)对硫磺和二硫化物的还原起重要作用，但该酶实际还原硫的形态(S)尚不清楚。在这里，我们测定了从平菇到27埃分辨率的含有FAD和辅酶A的全酶的结构，并表征了它的底物特异性。这种酶是相对混杂的，可以还原一系列的二硫化物、过硫化物和多硫化物。这些结果表明，在体内可能的底物是辅酶A的NAD(P)H和二、多硫化物和过硫化物的衍生物，尽管多硫化物本身也被有效地还原。然而，这些结果并不排除该酶在单质硫(S-8)原位还原中的作用，并讨论了该底物可能的作用。在好氧过硫化物还原过程中，观察到过硫化物底物的快速循环，这可能是通过O-2和/或H_2O_2氧化硫化物而发生的。正如预期的那样，这种反应在厌氧条件下消失，并可能解释其他人的观察结果，即Coadr对S-0的呼吸不是必需的，但在S-0存在的情况下似乎参与了氧化防御。与来自Shewanella loihica PV-4的同源Npsr酶和已知能还原CoA二硫化物的同源酶相比，phCoADR结构显示了一个相对受限的底物通道，通向FAD异四氧嘧啶环的硫侧，这表明这一酶类可能会选择特定的二硫化物底物。
FAD and NAD(P)H-dependent coenzyme A disulfide reductases/polysulfide reductases (CoADR/Psr) have been proposed to be important for the reduction of sulfur and disulfides in the sulfur reducing anaerobic hyperthermophiles Pyrococcus horikoshii and Pyrococcus furiosus; however, the form(s) of sulfur that the enzyme actually reduces are not clear. Here we determined the structure for the FAD- and coenzyme A-containing holoenzyme from P. horikoshii to 27 angstrom resolution and characterized its substrate specificity. The enzyme is relatively promiscuous and reduces a range of disulfide, persulfide, and polysulfide compounds These results indicate that the likely in vivo substrates are NAD(P)H and di-, poly-, and persulfide derivatives of coenzyme A, although polysulfide itself is also efficiently reduced. The role of the enzyme in the reduction of elemental sulfur (S-8) in situ is not, however, ruled out by these results, and the possible roles of this substrate are discussed. During aerobic persulfide reduction, rapid recycling of the persulfide substrate was observed, which is proposed to occur via sulfide oxidation by O-2 and/or H2O2. As expected, this reaction disappears under anaerobic conditions and may explain observations by others that CoADR is not essential for S-0 respiration in Pyrococcus or Thermococcus but appears to participate in oxidative defense in the presence of S-0. When compared to the homologous Npsr enzyme from Shewanella loihica PV-4 and homologous enzymes known to reduce CoA disulfide, the phCoADR structure shows a relatively restricted substrate channel leading into the sulfur-reducing side of the FAD isoalloxazine ring, suggesting how this enzyme class may select for specific disulfide substrates.