Transketolase catalyses asymmetric carbon-carbon bond formation and produces 1,3-dihydroxyketones, a functionality that is found in a vast number of natural and synthetic compounds. The wild-type transketolase enzymes from several species can accept a wide range of aldehydes, but industrially exploitable levels of activity tend to be limited to natural substrates and small aliphatic aldehydes. Several single mutants of the transketolase from E. coli were found to have enhanced activity towards non-phosphorylated substrates, non-hydroxylated aldehydes, and cyclic aldehydes. However, aromatic aldehydes still suffer from poor activities and yields while the key bottlenecks have not been identified. The strategy for creating new libraries from combining single mutations can have significant impact not only on the activity but also the stability of the enzyme due to the synergy between residues. On the other hand, the combination of two sites identified within a co-evolved network has created mutants with high towards propionaldehyde and decent stability. Kinetic studies of a small transkeloase library with 3-formylbenzoic acid (3-FBA) and 4-formylbenzoic acid (4-FBA) suggested that the affinity between the enzyme and the aromatic aldehyde, as well as their proximal orientations, was the key factor governing the reaction rate. This was also supported by computational modelling of substrate binding. Site-saturation mutagenesis at S385 and R358 was performed to further improve the activity of transketolase for 3-FBA, 4-FBA and also 3-hydroxybenzaldehyde (3-HBA). The new mutants were then assessed alongside transaminase for the ability to synthesise novel aromatic amino alcohols, which would provide building blocks for chloramphenicol and its derivatives. However, none of the available transaminases appeared to accept either of the compounds. The competitive reaction between 4-FBA and 4-DOPBA, the dihydroxy ketone product of 4-FBA, in an amination reaction suggested that 4-DOPBA was unable to access into the active site of CV2025 transaminase.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626738 |
| Date | January 2014 |
| Creators | Payongsri, P. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1417375/ |
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