The use of enzymes as catalysts for chemical transformations has emerged as a “greener” alternative to traditional organic synthesis. An issue to solve though, is that enzymes are designed by nature to catalyze reactions in a living cell and therefore, in many cases, do not meet the requirements of a suitable biocatalyst. By mimicking Darwinian evolution these problems can be addressed in vitro by different types of directed evolution strategies. α-Hydroxy aldehydes and α-hydroxy ketones are important building blocks in the synthesis of natural products, fine chemicals and pharmaceuticals. In this thesis, two alcohol dehydrogenases, FucO and ADH-A, have been studied. Their potentials to serve as useful biocatalysts for the production of these classes of molecules have been investigated, and shown to be good. FucO for its strict regiospecificity towards primary alcohols and that it strongly prefers the S-enantiomer of diol substrates. ADH-A for its regiospecificity towards secondary alcohols, its enantioselectivity and that is has the ability to use a wide variety of bulky substrates. The kinetic mechanisms of these enzymes were investigated using pre-steady state kinetics, product inhibition, kinetic isotope effects and solvent viscosity effects, and in both cases, the rate limiting steps were pin-pointed to conformational changes occurring at the enzyme-nucleotide complex state. These characterizations provide an important foundation for further studies on these two enzymes. FucO is specialized for activity with small aliphatic substrates but is virtually inactive with aryl-substituted compounds. By the use of iterative saturation mutagenesis, FucO was re-engineered and several enzyme variants active with S-3-phenylpropane-1,2-diol and phenylacetaldehyde were obtained. It was shown that these variants capability to act on larger substrates are mainly due to an enlargement of the active site cavity. Furthermore, several amino acids which are important for catalysis and specificity were identified. Phe254 interacts with aryl-substituted substrates through π-π stacking and may be essential for activity with these larger substrates. One mutation caused a loss in the interactions made between the enzyme and the nucleotide and thereby enhanced the turnover number for the preferred substrate
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-208139 |
| Date | January 2013 |
| Creators | Blikstad, Cecilia |
| Publisher | Uppsala universitet, Institutionen för kemi - BMC, Uppsala |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 ; 1081 |
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