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Title: The application of mutagenesis to explore the role of primary structure in the function of a range of enzymes in E. coli
Author: Al-Shamarti, Mohammed Jasim Mohammed
ISNI:       0000 0004 5992 6182
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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It has become possible over the last decade to compare the primary structures of proteins from many hundreds of species at the push of a button. This rich source of information on the relationship between polypeptide sequence and function is however limited since there is often no experimental evidence for protein activity. By amplifying open reading frames using an error prone DNA polymerase, I have combined the power of comparative BLAST analysis with functional screening in order to “stress test” the relationships between primary structure and function in a number of “model” enzymes which include M.HhaI 5-cytosine methyltransferase, chloramphenicol acetyltransferase (CATI), and dihydrolipoamide dehydrogenase (E3). These enzymes models represent quite different structural and functional classes, and to strengthen any general conclusions, they have been chosen here to make the evaluation of any impact on changes in primary structure on enzyme activity, as comprehensive as possible. The results showed different patterns of tolerance toward the random primary structure changes. Dihydrolipoamide dehydrogenase appeared to be the most resilient enzyme, followed by M.HhaI and CATI respectively. This suggests that enzymes of different structural and functional types will have different degrees of tolerance and/or flexibility (in term of activity maintenance) in accommodating primary structure changes. Comparing the present mutagenesis and activity screening data with the available biochemical data of the related enzymes, as well as plotting the positions of mutations on the relevant secondary and three dimensional structures, showed interesting traits in all enzymes to tolerate primary structure changes even in regions of the enzymes normally considered to be sensitive points, while some amino acid residues of no previously assigned importance could abolish enzyme activity completely.
Supervisor: Hornby, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available