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Abstract：
Redox-dependent thiol-disulfide switches of cysteine residues are one of the significant posttranslational modifications of proteins to control rapidly their stability, activity, and protein interaction. Redox control also modulates the tetrapyrrole biosynthesis (TBS). Among the redox-dependent TBS enzymes, 5-aminolevulinic acid dehydratase (ALAD) was previously recognized to interact with reductants, such a thioredoxins or NADPH-dependent thioredoxin reductase C. In this report, we aim to verify the redox sensitivity of ALAD and identify the redox-reactive cysteine residues among the six cysteines of the mature protein form Arabidopsis. Based on structural modelling and comparative studies of wild-type ALAD and ALAD mutants with single and double Cys➔Ser substitutions under oxidizing and reducing conditions, we aim to predict the dimerization and oligomerisation of ALAD as well as the crucial Cys residues for disulfide bridge formation and enzyme activity. The Cys404Ser mutation led to a drastic inactivation of ALAD and redox-dependent properties of ALAD were severely impaired, when Cys71 was simultaneously mutated with Cys152 or Cys251. Cys71 is located in a flexible N-terminal arm of ALAD, which could allow intramolecular disulfide bridges with Cys residues at the surface of the remaining globule ALAD structure. As a result, we propose different roles of Cys residues for redox control, catalytic activity and Mg2+-dependent assembly.
摘要：
半胱氨酸残基的氧化还原依赖性巯基-二硫键开关是蛋白质的重要翻译后修饰之一，可以快速控制其稳定性。活动,和蛋白质相互作用。氧化还原控制还调节四吡咯生物合成(TBS)。在氧化还原依赖性TBS酶中，5-氨基乙酰丙酸脱水酶（ALAD）以前被认为与还原剂相互作用，例如硫氧还蛋白或NADPH依赖性硫氧还蛋白还原酶C。在本报告中，我们的目的是验证ALAD的氧化还原敏感性，并在拟南芥成熟蛋白的六个半胱氨酸中鉴定氧化还原反应性半胱氨酸残基。基于在氧化和还原条件下具有单Cys和双Cys的野生型ALAD和ALAD突变体的结构建模和比较研究，我们旨在预测ALAD的二聚化和寡聚化以及二硫键形成和酶活性的关键Cys残基。Cys404Ser突变导致ALAD严重失活，ALAD的氧化还原依赖性特性严重受损，当Cys71与Cys152或Cys251同时突变时。Cys71位于ALAD的柔性N端臂中，这可以允许分子内二硫键与Cys残基在剩余的球ALAD结构的表面。因此,我们提出了Cys残基在氧化还原控制中的不同作用，催化活性和Mg2+依赖组装。
