Structure, Mechanism, and Physiology of Two Sulfur-Mobilizing Enzymes from Synechocystis 6803

含有铁硫（Fe-S）簇的蛋白质对所有生物体都是必不可少的。最近，在定义生物学中这些复杂集群的组装机制方面取得了重大进展。所谓的Isc Fe-S组装系统的组件在生命的所有三个领域都很普遍。Isc机器的中心齿轮是吡哆醛磷酸依赖的半胱氨酸脱硫酶IscS。在Fe-S合成/插入过程中，多个辅助蛋白与IscS相互作用和合作。最近在细菌和植物中发现了一个被称为Suf的独特系统，它很可能是植物叶绿体的主要组装装置。类似于IscS的蛋白质（SufS）很可能是Suf组装系统的等效硫贩运者。与SufS合作的其他辅助蛋白尚未确定，这些因子的发现是本研究的一个方面。据推测，SufS脱硫酶的机制与更广泛表征的IscS脱硫酶相似，但Bollinger实验室的初步数据表明，可能存在重要差异。该项目将阐明SufS反应的机理及其与IscS反应的不同程度。有证据表明，Suf系统可能是蓝藻中Fe-S组装的主要原因。另外，一种类似的蛋白质（囊肿(e)线C-S裂解酶或C-DES）通过一种独特的机制从半胱氨酸及其s取代衍生物中裂解硫，可能是关键的硫动员铁-s组装因子。该项目将确定哪些蛋白质在蓝藻铁硫合成中是最重要的，如果两者都重要，或者如果两者都不重要。半胱氨酸脱硫酶的机理表征产生了详细的假设，以解释脱硫酶如何避免催化C-DES促进的硫消除反应，反过来，C-DES如何实现对半胱氨酸的特异性，而有利于S取代衍生物，如胱氨酸和S-（烷基）半胱氨酸。基于这些假设的预测将被测试，以评估这些类似酶的功能差异背后的分子逻辑。自然界的复杂程度远远超过我们人类的两个化学领域是：(1)作为催化剂活性中心的复杂无机组合的构建；(2)催化剂（酶）的设计，在给定的底物可能存在的一系列化学相似途径中，只促进一种反应。人们期望，对大自然在这两个领域的策略的更深刻的理解将激发新的化学过程的发展。这项研究旨在更好地理解自然界的多种策略，以合成一种特别通用和重要的复杂无机组合，铁硫簇。由于铁硫团簇在固氮、呼吸和光合作用等生化过程中起着重要作用，预计该结果有朝一日可能会启发重要的仿生催化剂的合成过程。作为了解生物铁硫簇组装的一部分，该项目将确定两种相似的酶如何在组装过程中动员所需的无机硫化物中发挥作用，从而选择两种化学上不同的反应途径来分解共同的底物半胱氨酸。将每种酶的结构与其促进的反应途径联系起来的原理将构成一个范例，说明如何通过对催化剂结构的微妙调整来实现化学特异性。

Proteins that contain iron-sulfur (Fe-S) clusters are essential to all organisms. Great progress has been made recently in defining the mechanisms by which these complex clusters are assembled in biology. Components of the so-called Isc Fe-S assembly system are widespread in all three domains of life. The central cog in the Isc machine is a pyridoxal phosphate-dependent cysteine desulfurase, IscS. Multiple accessory proteins interact and cooperate with IscS in Fe-S synthesis/insertion. A distinct system, designated as Suf, has very recently been recognized in bacteria and plants and is likely to be the primary assembly apparatus in plant chloroplasts. A protein (SufS) similar to IscS is likely to be the equivalent sulfur-trafficker for the Suf assembly system. Additional accessory proteins that cooperate with SufS have not yet been identified, and discovery of these factors is one aspect of this research. It has been presumed that the mechanism of the SufS desulfurase is similar to that of the more extensively characterized IscS desulfurase, but preliminary data from the Bollinger lab suggest that there may be important differences. The project will elucidate the mechanism of the SufS reaction and the extent to which it differs from that of IscS. Evidence suggests that the Suf system may be primarily responsible for Fe-S assembly in cyanobacteria. Alternatively, a similar protein (cyst(e)ine C-S lyase or C-DES) that cleaves sulfur from cysteine and its S-substituted derivatives by a distinct mechanism may be the key sulfur-mobilizing Fe-S assembly factor. The project will determine which of these proteins is of primary importance in cyanobacterial Fe-S synthesis, if both are important, or if neither is. Mechanistic characterization of cysteine desulfurases has engendered detailed hypotheses to explain how the desulfurases avoid catalyzing the sulfur elimination reaction promoted by C-DES and, conversely, how C-DES achieves specificity against cysteine in favor of S-substituted derivatives such as cystine and S-(alkyl)cysteines. Predictions based on these hypotheses will be tested in order to evaluate the molecular logic underlying the functional divergence of these similar enzymes. Two areas of chemistry in which Nature's sophistication far exceeds our own are (1) the construction of complex inorganic assemblies for use as the active centers of catalysts and (2) the design of catalysts (enzymes) that promote only one type of reaction among a range of chemically similar pathways that may be available with a given substrate. It is expected that a more profound understanding of Nature's strategies in these two areas would inspire development of new chemical processes. This research seeks a better understanding of Nature's multiple strategies for synthesis of one particularly versatile and important class of complex inorganic assembly, the iron-sulfur clusters. As iron-sulfur clusters are important in biochemical processes ranging from nitrogen fixation to respiration to photosynthesis, it is expected that the results may one day inspire synthetic processes for construction of important biomimetic catalysts. As part of the effort to understand biological iron-sulfur cluster assembly, the project will determine how two similar enzymes that may each have a role in mobilizing the inorganic sulfide needed for the assembly process can select two chemically distinct reaction pathways for breakdown of a common substrate, cysteine. The principles relating the structure of each enzyme to the reaction pathway it promotes will constitute a paradigm for how chemical specificity may be achieved by subtle tuning of catalyst structure.