PDF(Pubmed)
Abstract：
In mammals, l-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (Mus musculus CDO numbering) and a nearby tyrosine at position 157 (Cys-Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys-Tyr cross-link during repeated enzyme turnover increases CDO\'s catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys-Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O2, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.
摘要：
在哺乳动物中,1-半胱氨酸（Cys）稳态由单核非血红素铁酶半胱氨酸双加氧酶（CDO）维持，将Cys氧化为半胱氨酸亚磺酸。CDO包含一种罕见的翻译后修饰，涉及在位置93的Cys残基（MusmusculusCDO编号）和位置157的附近酪氨酸（Cys-Tyr交联）之间形成硫醚交联。分离的CDO包含交联和非交联的同工型，在重复的酶周转过程中，Cys-Tyr交联的形成使CDO的催化效率提高了~10倍。有趣的是,虽然C93GCDO变体缺乏Cys-Tyr交联，它与交联的野生型(WT)CDO具有类似的活性。或者，Y157FCDO变体，它也缺乏交联，但在93位保持游离硫醇盐，表现出急剧降低的催化效率。这些观察表明C93的未束缚的硫醇盐部分对CDO活性有害和/或Y157对于催化是必需的。为了进一步评估残基C93和Y157的作用,我们对Y157FCDO和新设计的C93G/Y157FCDO变体进行了光谱和动力学表征。我们的结果提供了证据，表明非交联的C93硫醇盐在Cys结合的Y157FFe（II）CDO的第六个配位位点稳定了水。水也存在，虽然协调较弱，在Cys结合的C93G/Y157FFe(II)CDO中。水分子的存在，必须被共底物O2取代，可能对Y157F和C93G/Y157FCDO变体的~15倍和~7倍降低的催化效率做出重大贡献，分别,相对于交联的WTCDO。
