Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626739
Title: Evolutionary and mutagenesis studies of E. coli transketolase to guide further protein engineering
Author: Rahimi Fard Jahromi, R.
ISNI:       0000 0004 5363 3151
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
Availability of Full Text:
Access through EThOS:
Full text unavailable from EThOS. Please try the link below.
Access through Institution:
Abstract:
The enzyme Escherichia coli (E. coli) transketolase (TK; E.C. 2.2.1.1) occupies a pivotal place in metabolic regulation. This enzyme catalyses the interconversion of sugars by transferring a two-carbon ketol unit from a ketose donor substrate to an aldolase acceptor substrate. It is also an important biocatalyst in stereo-specific carbon-carbon bond synthesis with potential industrial application for the synthesis of pharmaceuticals, agrochemicals and fine chemicals. Although many useful reactions have been reported for TK, many of the substrates and products are unstable or insoluble at the pH or temperature for which the enzyme has optimum activity. Understanding the structural stability of transketolase using mutagenesis under bioprocess conditions will improve our capacity to comprehend and ultimately to engineer it to make it work in a broader range of pH or temperature to potentially help in the reduction of process time and to increase the quality and solubility of products. In this thesis, Chapter 3 studied the denaturation of E. coli TK at extreme temperature and pH. DLS data for thermal study of holo-TK at pH 7 indicated that aggregation occurred at above 58 °C using a fixed temperature ramping rate. However, the thermal denaturation profile measured by CD for holo-TK showed a gradual non-cooperative loss of secondary structure as the temperature increased between 5 and 50 °C, and the mid-point of unfolding/aggregation under the same conditions was confirmed to occur at 58.3 °C. The enzyme was observed to be more tolerant at high pH, both structurally and catalytically, where no loss of tertiary structure was evident at pH 9 and 50% of enzyme activity was retained at pH 11. All the results presented in this chapter were published in Jahromi et al. 2011 which provide a good and solid base for protein engineering of the E. coli TK. Chapter 4 investigated the impact of dimer interface mutations on the thermal stability of the E. coli TK. The aim of this Chapter was to determine regions of the dimer interface that were more susceptible to aggregation. Mutations in this study 4 were limited to the dimer interface region and at least at a 4Å distance from the active-site. Computational tools such as Aggrescan, Tango and Pasta were used to predict the aggregation prone regions in the structure. None of the mutants in this study showed a higher specific activity compared to the wild-type as expected. The denaturation profiles from CD and DLS measurements showed mutants E647Q and D617N to have higher unfolding/aggregation temperatures compared to wild-type. The double mutants H94A-E647Q and D617N-K621M were shown to have additive effects compared to their corresponding single mutants. This study revealed that mutations of the PP-domain and Pyr-domain of E. coli TK are more prone to aggregation. This could be explained by the fact that the residues in these two domains are the ones that mainly contribute to the dimer interface stability. Chapter 5 studies mutagenesis of E. coli transketolase using consensus concept. Mutations were spread in all three domains and located either at the dimer interface or buried in the structure. This study revealed remarkable results with all of the seven mutants having stabilising effect on the enzyme compared to wild type. Previous studies have reported this technique to identify only 30%-50% of the mutants to be stabilising. The result of temperature ramping on the secondary structure of the consensus mutants, as measured by CD, showed that all of the mutants apart from F554L, displayed a stabilising effect, with an increase in the mid-point of unfolding/aggregation temperature in the range of 0.3°C to 4.3°C. Interestingly, F554L also had higher CD and DLS thermal unfolding/aggregation onset points compared to wild-type TK. The I365L mutant showed the highest increase in the mid-point of unfolding/aggregation of about 4.3 °C. Another remarkable result was that the I365L mutant also showed the highest specific activity retained both in purified and lysate form which were approximately 67% and 20% respectively. G506A was another interesting mutant that showed above 50% specific activity retained in the purified form. Overall, the consensus sequence approach has proven to be the most effective technique for generating enzyme variants with improved aggregation propensity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.626739  DOI: Not available
Share: