Rewiring P450s for efficient biocatalysis

该项目将新型P450催化剂与尖端酶工程技术相结合，以选择性地羟基化底物分子。P450在精细化学品（香料、调味品、药品）和日用化学品（燃料、溶剂、农用化学品）的可持续生产方面具有巨大的潜力，但受到其机制中关键电子转移步骤的限制。在这个项目中，将设计新的融合蛋白，其中血红素和还原酶结构域之间的电子转移途径被优化，以最大限度地提高催化效率。新的“重新布线”P450然后将通过最先进的电子顺磁共振（EPR）光谱进行研究。在这里，两个自旋活性物质（无论是天然的蛋白质，或添加使用氮氧自旋标记）将被纳入和使用EPR在催化相关的条件下获得位于还原酶和血红素域的这些标签之间的纳米级距离测量。通过计算技术的进一步研究将使这些发现合理化，从而充分了解P450如何重新布线，以最大限度地提高其在工业应用中的效用。

This project combines novel P450 catalysts with cutting edge enzyme engineering techniques to selectively hydroxylate substrate molecules. P450s have enormous potential for the sustainable manufacture of both fine chemicals (fragrances, flavourings, pharmaceuticals) and commodity chemicals (fuels, solvents, agrochemicals) but are limited by key electron transfer steps in their mechanisms. In this project, new fusion proteins will be engineered, in which electron transfer pathways between haem and reductase domains are optimised to maximise catalytic efficiency. The new "re-wired" P450s will then be investigated by state of the art electron paramagnetic resonance (EPR) spectroscopy. Here, two spin active species (either native to the protein, or added using nitroxide spin labels) will be incorporated and used to obtain nanoscale distance measurements between these labels situated in the reductase and haem domains using EPR under catalytically relevant conditions. Further study by computational techniques will rationalise the findings and thereby provide a full understanding of how P450s can be rewired to maximise their utility for industrial applications.