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

集胞藻 6803 中两种硫动员酶的结构、机制和生理学

• 批准号: 0235979
• 负责人: Joseph Bollinger
• 金额: $ 44.6万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2006-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0235979&HistoricalAwards=false
• 关键词: Structure; Mechanism; Physiology; Two; Sulfur

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.

含有铁-硫簇(Fe-S)的蛋白质是所有生物体所必需的。最近在确定这些复杂簇在生物学中组装的机制方面取得了很大进展。所谓的ISC Fe-S组装系统的组件广泛存在于生活的所有三个领域。ISC机器的中心齿轮是一种依赖于磷酸吡哆醛的半胱氨酸脱硫酶，ISCS。在铁-S的合成/插入过程中，多种辅助蛋白与ISCs相互作用和协同作用。一个独特的系统，命名为Suf，最近在细菌和植物中被发现，很可能是植物叶绿体中的主要组装装置。一个类似于ISCS的蛋白质(SufS)很可能是Suf组装系统的等效硫运输者。其他与SufS协同作用的辅助蛋白还没有被鉴定出来，这些因子的发现是本研究的一个方面。据推测，SufS脱硫酶的机制类似于特征更广泛的ISCS脱硫酶，但来自布林格实验室的初步数据表明，可能存在重要的差异。该项目将阐明SuFS反应的机理以及它与ISCS反应的不同程度。有证据表明，SuF系统可能是蓝藻体内Fe-S组装的主要原因。或者，一种类似的蛋白质(胞苷C-S裂解酶或C-DES)通过不同的机制从半胱氨酸及其S取代的衍生物中分解硫，可能是关键的硫动员铁-S组装因子。该项目将确定这些蛋白质中的哪一种在蓝藻的铁-S合成中起主要作用，如果两者都是重要的，或者都不是。半胱氨酸脱硫酶的机理表征已经产生了详细的假说来解释脱硫酶是如何避免催化C-DES促进的硫消除反应的，反过来，C-DES如何实现对半胱氨酸的专一性，有利于S取代的半胱氨酸和S-(烷基)半胱氨酸的衍生物。基于这些假设的预测将得到检验，以评估这些类似酶功能差异背后的分子逻辑。自然界在两个化学领域的复杂性远远超过我们自己的：(1)构建复杂的无机组件，用作催化剂的活性中心；(2)催化剂(酶)的设计，在给定底物可能存在的一系列化学相似的途径中，仅促进一种类型的反应。预计对自然界在这两个领域的战略有更深刻的理解将激励新的化学过程的发展。这项研究试图更好地理解自然界合成一种特别多功能和重要的复杂无机组装--铁-硫簇--的多种策略。由于铁-硫团簇在从固氮到呼吸再到光合作用的各种生化过程中都很重要，预计有一天这一结果可能会启发合成工艺来构建重要的仿生催化剂。作为了解生物铁-硫簇组装工作的一部分，该项目将确定两种类似的酶，它们各自可能在动员组装过程所需的无机硫化物方面发挥作用，它们如何选择两条化学上不同的反应路径来分解共同的底物半胱氨酸。将每种酶的结构与它所促进的反应途径联系起来的原则将构成一个范例，说明如何通过微妙地调整催化剂结构来实现化学专一性。