In Vitro Evolution of Aldolases; Towards a Baylis-Hillmanase

  • Michael Swiatyj

Student thesis: Phd


The fructose 1,6-bisphosphate type I aldolase from the thermophilic organism Thermoproteus tenax has been expressed and purified by heat treatment. The aldolase aldehyde substrate scope was investigated using electrospray mass spectrometry to detect the formation of any aldol products. Parameters affecting aldolase activity, including temperature, buffer pH and solvent additive were investigated.The synthesis of an aldehyde with an attached fluorescent reporter group was performed for potential use in the screening of mutant aldolases for aldol or Baylis-Hillman activity. The synthesis of 1-hydroxy-3-buten-2-one phosphate, an analogue of dihydroxyacetone phosphate, capable of participating in Baylis-Hillman reactions, was achieved in 5 steps from 3-buten-1-ol. This analogue was used in the investigation of the wild type aldolase and several mutant aldolases for Baylis-Hillman activity.X-ray crystallographic data was obtained for the wild type enzyme and the Trp144Leu mutant aldolase with 1-hydroxy-3-buten-2-one phosphate bound at their active sites. In the wild type aldolase, the substrate was found to bind in a similar manner to dihydroxyacetone phosphate, with the formation of a Schiff base with the Lys177 amino acid residue at the enzyme active site. In the Trp144Leu mutant aldolase, Lys177 has added in Michael fashion to the enone functionality of the bound substrate forming an enolate instead of forming a Schiff base. Both forms of the bound substrate are potentially capable of participating in Baylis-Hillman reactions. The enzymes have yet to be fully investigated for Baylis-Hillman activity.
Date of Award1 Aug 2012
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDavid Berrisford (Supervisor)


  • Aldolase
  • Baylis-Hillman

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