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Title: Enhancing the promiscuity of sugar kinases
Author: Kristiansson, Helena
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2012
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Abstract:
Glycosides play a major part in the development of novel pharmaceuticals since the sugar ligands of natural products play important roles in, e.g., cell-wall synthesis. The use of phosphosugars in the pharmaceutical industry has been linked to anti-inflammatory and immunosuppressive drugs. Therefore it's of interest to be able to develop and manufacture unique glycosylated natural products and investigate their abilities as potential drugs. A technique called In Vitro Glycorandomization (IVG) has been developed for the production of novel varieties of the glycopeptide antibiotic vancomycin. IVG need a range of sugar-l-phosphates for the production of different NDP-sugars. An enzyme that can phosphorylate a wide range of sugar substrates at position 1 is of importance to simplify the technique. Two human enzymes, galactokinase and N-acetylgalactosamine kinase, were mutated at specific residues in their amino acid sequence in an attempt to increase the number of sugars each enzyme could phosphorylate. Galactokinase showed activity toward two sugars not including its natural substrate galactose: 2-deoxy-D-galactose and D-galactosamine-hydrochloride. The promiscuity of galactokinase was increased after mutating the tyrosine at position 379. The Y379W mutant was the most successful by phosphorylating seven of the nine sugars selected for this enzyme. Several N-acetylgalactosamine kinase mutants showed higher activity toward the natural substrate (N-acetylgalactosamine) but, overall, the wild type was the better enzyme for the three selected sugars for this enzyme. Since no structural changes were visible when performing homology modelling of the mutants it is suggested that the increase in promiscuity for galactokinase is due to a rearrangement of hydrogen bonds within the proteins. It was shown that the flexibility of the galactokinase enzymes declines when substrates are bound in the active site and the mutants became more rigid than the wild type. The mutants are, however, more flexible than the wild type before the substrates bind.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.579758  DOI: Not available
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