AI Article Synopsis
- The enzyme 4-oxalocrotonate tautomerase (4-OT) aids in breaking down aromatic hydrocarbons and can also catalyze various carbon-carbon bond-forming reactions, like the condensation of acetaldehyde and benzaldehyde to form cinnamaldehyde.
- Researchers improved the efficiency of 4-OT as an aldolase through engineering, achieving a greater than 5000-fold increase in catalytic efficiency and over 10 million-fold increase in reaction specificity.
- The study involved systematic mutation of the enzyme, identifying "hotspots" near the active site that, when modified, significantly enhanced the desired aldolase activity.
Article Abstract
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.
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| http://dx.doi.org/10.1002/cbic.201600149 | DOI Listing |
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