Long Range Control of Reactivity in CO Dehydrogenases

同源酶是由一组高度保守的“核心”残基定义的，这些残基是功能所必需的。催化性能（催化速率，米切里斯常数，对抑制剂的敏感性，甚至双向性）通常由远离活性位点的非保守残基决定。识别核心残基、定义功能、了解次级球残基的影响以及理解远端效应是揭示金属酶的机制和理解它们如何被进化调节的基础。我们的建议涉及NiFe CO脱氢酶（CODHs），它催化CO2可逆还原为CO。合作伙伴（CNRS和HU）分别或共同为阐明CODHs的机制做出了贡献。HU最近分离并表征了一种特殊的酶，CooS-V，它在序列上与原型CODHs非常相似，但执行一个独特的（尚未未知的）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中功能重要的构象变化。CooS-V是最接近coh的酶，但又不是coh，因此它提供了一个机会来了解定义coh功能的核心残基，以及通过位点定向诱变将coh功能改造成CooS-V或将coh转化为具有CooS-V活性的酶来调节coh活性的远程相互作用。在这个项目中，双方合作伙伴将设计和生产介于已知CODHs和CooS-V之间的蛋白质变体，他们将联合使用晶体学（HU）和电化学（CNRS）来表征它们，提供结构信息和深入的功能和动力学表征。将双方的方法结合起来，将使人们对CODHs的不同特性如何演变以及如何用于未来的应用有前所未有的了解。

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.