Regulating the activity and selectivity of squalene-hopene-cyclases via the membraneanchor

通过膜锚调节角鲨烯-霍烯环化酶的活性和选择性

• 批准号: 524081117
• 负责人: Professor Dr. Bernhard Hauer
• 金额: --
• 依托单位: Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (ibwf)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/524081117?language=en
• 关键词: Regulating; activity; selectivity; squalene; hopene

Consideration of the complex membrane environment is great importance to the study of membrane-bound enzymes but leads to difficulties when studying the enzymes in isolation. Although new methods have increased the accessibility of membrane-bound enzymes as part of a complex multicomponent system, there is still an asymmetry in our knowledge to the detriment of these proteins. An example of this knowledge gap is the lack in our understanding of how membrane-bound enzymes are regulated by different membrane properties such as composition, geometry, or biochemical and physical factors. To elucidate a part of this interaction this project will investigate the membrane anchor of monotopic proteins as a possible regulatory element. As a model system to study this element of the enzymatic structure, we consider the monotopic triterpene cyclase squalene-hopene cyclase (SHC). These enzymes control membrane fluidity by producing highly hydrophobic hopanoids to counteract biological stress on the host organism. Therefore, a mutual influence of membrane fluidity and enzyme activity of SHCs seems comprehensible. Supporting this hypothesis we observed in previous studies, that the promiscuous cyclization of (E,E)-homofarnesol to (-)-ambrox in the in vivo system was subject to substrate-excess inhibition, which was not observed for the purified enzyme. Thus, it can be speculated that local membrane perturbations caused by terpenes, result in an inhibitory effect on the activity of SHCs. To investigate the role of the anchor motif in this interaction, we plan to generate a focused collection of rational SHC variants whose modifications focus on this motif and the surrounding amino acids. These variants will first be selected for indications of alterations in membrane interaction using a series of model reactions and subsequently characterized by introduction into E. coli or mycoplasmas with modified membrane composition. The aim is to investigate whether the modification of the membrane anchor affects or even completely uncouples the interaction between the membrane and enzymatic activity, thus identifying the membrane anchor as a mediator between membrane and catalytic apparatus. Indeed, prior reconstruction of the Free Energy Landscape of the wildtype and a laboratory-evolved variant showed that different conformations of this α-helix could be displayed by the different SHC variants when they are embedded on the lipid membrane. To support these studies, in addition to characterization in the laboratory, further computational studies of the generated variants will also be performed taking into account varying membrane system to rationalize the influence of the introduced mutations. A deeper understanding of these relationships would add another element to our understanding of membrane proteins and could allow us to draw conclusions on the regulation of other monotopic membrane proteins.

考虑复杂的膜环境对膜结合酶的研究具有重要意义，但也给酶的分离研究带来了困难。尽管新的方法增加了膜结合酶作为复杂多组分系统的一部分的可及性，但我们对这些蛋白质的认识仍然不对称，这对这些蛋白质不利。这种知识鸿沟的一个例子是，我们缺乏对膜结合酶如何受到不同膜性质(如组成、几何形状或生化和物理因素)调节的了解。为了阐明这种相互作用的一部分，该项目将研究单调蛋白质的膜锚作为可能的调节元件。作为研究酶结构中这一元件的模型系统，我们考虑了单调三萜环化酶角鲨烯-Hopene环化酶(SHC)。这些酶通过产生高度疏水的类胡萝卜素来控制膜的流动性，以抵消对宿主生物体的生物压力。因此，SHCS的膜流动性和酶活性的相互影响似乎是可以理解的。支持我们在以前的研究中观察到的这一假说，在体内系统中，(E，E)-高阿糖醇到(-)-ambrox的混杂环化受到底物过量抑制，而纯化的酶没有观察到这一点。因此，可以推测，由萜类引起的局部膜扰动对SHCS的活性具有抑制作用。为了研究锚定基序在这种相互作用中的作用，我们计划产生一个有针对性的合理SHC变体的集合，其修改集中在该基序和周围的氨基酸上。这些变异体将首先通过一系列模型反应来选择膜相互作用变化的迹象，然后通过引入具有改良膜成分的大肠杆菌或支原体来表征。目的是研究膜锚的修饰是否影响甚至完全解偶联膜与酶活性之间的相互作用，从而确定膜锚是膜和催化装置之间的中介。事实上，先前对野生型和实验室进化变体的自由能景观的重建表明，当不同的α变体嵌入脂膜时，它们可以显示出不同的构象。为了支持这些研究，除了在实验室中进行表征外，还将对产生的变异体进行进一步的计算研究，同时考虑到不同的膜系统，以使引入的突变的影响合理化。对这些关系的深入理解将为我们对膜蛋白的理解增加另一个元素，并使我们能够得出关于其他单位膜蛋白调控的结论。