Rahimi Mehran, van der Meer Jan-Ytzen, Geertsema Edzard M, Poddar Harshwardhan, Baas Bert-Jan, Poelarends Gerrit J
Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.
Institute for Life Science & Technology, Hanze University of Applied Sciences, Zernikeplein 11, 9747, AS, Groningen, The Netherlands.
Chembiochem. 2016 Jul 1;17(13):1225-8. doi: 10.1002/cbic.201600149. Epub 2016 May 30.
The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.
4-草酰巴豆酸互变异构酶（4-OT）作为芳烃降解途径的一部分，催化烯醇-酮互变异构，同时也能杂乱地催化各种碳-碳键形成反应。这些反应包括乙醛与苯甲醛的羟醛缩合反应生成肉桂醛。在此，我们证明，通过探索仅改变“热点”的小型文库，4-OT可被改造成为更高效的用于该缩合反应的醛缩酶，其催化效率（kcat /Km）提高了5000倍以上，反应特异性变化超过10^7倍。通过系统诱变（覆盖每个残基）确定热点，随后筛选能显著提高所需醛缩酶活性的单突变。所有有益突变都靠近4-OT的活性位点，从而支持了这样一种观点，即通过靠近活性位点的突变能更有效地增强杂乱酶的新催化活性。
