English (United States)
As fossil fuel reserves deplete, biofuels are becoming an increasingly important alternative energy source. Lignin, a paper industry waste product, is the most abundant aromatic compound found in nature and the products of its degradation by enzymes could be utilized in the generation of biofuels. DesB, DesZ, and LigAB are extradiol dioxygenase enzymes of the Protocatechuate Dioxygenase Superfamily, which are involved in the lignin degradation pathway of Sphingobium sp. SYK-6. Dioxygenase enzymes play a vital role in the metabolism of small aromatic molecules by cleaving the aromatic ring and allowing the molecules to be funneled into the TCA cycle. In earlier studies, DesB was shown to have strict specificity for its natural substrate gallate. This project has supported this conclusion, as DesB was not active when tested with a number of alternative substrates. A number of inhibitors have been identified, the most potent of which is is Protocatechuate (PCA). DesZ, the second extradiol dioxygenase investigated, was successfully expressed however not active during attempted kinetics assays. Work done previously by the Taylor lab has led to the discovery of heteroallosteric activation of LigAB by vanillin. This project explored the effects of mutating residues located in the putative allosteric binding site. Kinetics assays, following mutagenesis of an alanine residue at position 18 to a tryptophan, support that Ala18β is located in the putative allosteric binding pocket. Future studies will further investigate the mechanism of vanillin heteroallosteric activation of LigAB.
Cohn, Erin Frances, "Exploring Lignin Degradation: An Investigation of Three Extradiol Dioxygenase Enzymes" (2015). Honors Theses - All. 1473.
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