Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations

作者：Rahimi Mehran; van der Meer Jan Ytzen; Geertsema Edzard M; Poddar Harshwardhan; Baas Bert Jan; Poelarends Gerrit J

来源：ChemBioChem, 2016, 17(13): 1225-1228.

DOI：10.1002/cbic.201600149

• aldolase activity
• catalytic promiscuity
• mutagenesis
• oxalocrotonate tautomerase
• protein engineering

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 (k(cat)/K-m) 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.

• 出版日期2016-7-1