Long Range Control of Reactivity in CO Dehydrogenases

同源酶由一组高度保守的“核心”残基定义，这些残基是发挥功能所必需的。催化性质(催化速率、米氏常数、对抑制剂的敏感性，甚至是双向性)通常由远离活性中心的非保守残基决定。识别定义功能的核心残基，了解次级球状残基的影响，以及了解长程效应，对于揭示金属酶的机制和了解它们是如何被进化调节的是基础。我们的建议涉及NiFe CO脱氢酶(CODHs)，它催化二氧化碳可逆还原为CO。合作伙伴(CNRS和HU)分别和共同为阐明CODHs的机制作出了贡献。Hu最近分离并鉴定了一种特殊的酶，Coos-V，它在序列上与典型的CODH非常相似，但执行不同的(尚不清楚的)反应。观察到的Coos-V活性位点的灵活性使人想起CNRS研究的CODH中在功能上重要的构象变化。由于Coos-V是最接近CODH的酶而不是CODH，Coos-V提供了机会，通过使用定点突变将CODH功能改造成Coos-V或将CODH转化为具有Coos-V活性的酶，从而既可以了解定义CODH功能的核心残基，也可以了解调节CODH活性的远程相互作用。在该项目中，双方将设计和生产介于已知CODHs和CoOS-V之间的蛋白质变体，他们将联手使用结晶学(HU)和电化学(CNRs)对它们进行表征，提供结构信息以及深入的功能和动力学表征。将这两个合作伙伴的方法结合在一起，将使人们对CODH的不同性质如何演变以及如何为未来的应用提供前所未有的洞察力。

Homologous enzymes are defined by a set of highly conserved “core” residues that are required for function. The catalytic properties (catalytic rates, Michaelis constants, susceptibility to inhibitors, and even bidirectionality) are frequently determined by non-conserved residues remote from the active site. Identifying core residues defining the function, understanding the influence of the secondary sphere residues, and comprehending long-range effects are fundamental to reveal the mechanisms of metalloenzymes and understand how they have been tuned by Evolution. Our proposal concerns NiFe CO dehydrogenases (CODHs), which catalyze the reversible reduction of CO2 to CO. The partners (CNRS and HU) have contributed to elucidating the mechanisms of CODHs, separately and together. HU has recently isolated and characterized one particular enzyme, CooS-V, which is very similar in terms of sequence to prototypical CODHs but performs a distinct (yet unknown) reaction. The observed flexibility of the CooS-V active site is reminiscent of conformational changes that are functionally important in a CODH studied by CNRS. With its unique situation of being the closest enzyme to a CODH without being one, CooS-V provides opportunities to learn both about the core residues that define CODH function and about the long-range interactions that modulate CODH activity by using site-directed mutagenesis to either engineer CODH function into CooS-V or transform a CODH into an enzyme with CooS-V activity. In this project, both partners will design and produce protein variants that are intermediate between known CODHs and CooS-V, and they will join forces to characterize them using crystallography (HU) and electrochemistry (CNRS), providing structural information and in-depth functional and kinetic characterization. Combining the approaches of both partners will give unprecedented insight into how the different properties of CODHs have evolved and can be harnessed for future applications.