Protein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas mediterranea, have been shown to possess the latter. This study involves the characterization of two such proteins, lysine-?-oxidase (LodA) and glycine oxidase (GoxA). They have been reported to contain the same cysteine tryptophylquinone (CTQ) cofactor. Both require the co-expression of a second protein, LodB and GoxB respectively to generate matured CTQ containing active protein. Kinetic analysis of the reaction catalyzed by LodA showed that it followed the usual Michaelis-menten mode of interaction with its substrates. GoxA on the other hand exhibited allosteric cooperativity for its substrate glycine. This was attributed to the dimeric conformation of the wildtype GoxA based on size exclusion chromatographic studies. Mutagenesis study of amino acid residues based on the crystal structure of LodA and a homology model of GoxA, have given a detailed idea about their structure-function relationship. Kinetic studies on mutants of Tyr211 of LodA along with Lys530 present at the substrate channel, showed effects on both Km for the substrates and kcat for the reaction. As a result these residues have shown their involvement in forming a gate-like structure to control the to and fro movement of the substrate and products. Corresponding to this residue, the Phe237 of GoxA has proved to be important in maintaining the allostericity, by mediating the stable dimer formation. From the kinetic parameters, Cys448 of LodA was found to be responsible for substrate specificity and affinity. Whereas, mutants of His466 of GoxA that correspond to the Cys448 residue, were unable to yield CTQ containing active GoxA. On the other hand, Asp512 of LodA and Asp547 of GoxA that correspond to each other, have been implicated for their involvement in CTQ biogenesis. This study therefore highlights how even though this new pool of enzymes have great degrees of similarity in terms of the cofactors and conserved active site residues, there are major differences in the mechanism of the reaction that they catalyze which on a broader sense could influence the overall physiological importance of the enzyme in the biological system.
If this is your thesis or dissertation, and want to learn how to access it or for more information about readership statistics, contact us at STARS@ucf.edu
Doctor of Philosophy (Ph.D.)
College of Medicine
Burnett School of Biomedical Sciences
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Sehanobish, Esha, "Structural and functional characterization of enzymes of a novel group of tryptophylquinone cofactor containing oxidases" (2016). Electronic Theses and Dissertations. 5061.