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Protocatechuate 4,5-dioxygenase (LigAB) catalyzes dioxygenation of multiple lignin derived aromatic compounds-such as protocatechuate (PCA), gallate (GA) and 3-O-methyl gallate (3OMG)-with decreasing proficiency as the molecule size increases. We predicted that phenylalanine-103 of the ␣ subunit (Phe103␣) controls substrate specificity through interaction with the C5-funtionality of bound substrates, and mutagenesis would enhance GA and 3OMG catalysis. LigAB with Phe103␣ mutations (F103V, F103T and F103H) displayed enhanced catalytic efficiency for dioxygenation of 3OMG, with mutants displaying 12-to 31-fold increases in k app cat /K app m , making these mutant enzymes more active with 3OMG than its native dioxygenase (DesZ). The F103T and F103V point mutants also exhibited allosteric activation for the dioxygenation of PCA and GA, respectively, in the presence of vanillin, as previously observed for LigAB. The enhanced utilization of substrates by these mutants makes them potentially useful for efforts to develop engineered organisms that catabolize lignin into biofuels or fine chemicals.

opportunity to explore the wonderful world of scientific research, despite not knowing that I was a 'troubled child.' The past three years of research has not only taught me about enzymology and biofuels, but more importantly it has helped me discover myself in the most unexpected ways. Thank you for 'science', your constant guidance, patience and support, the shared tears and laughter, the life lessons and above all for believing in me. I would also like to thank grad student Kevin for being such a tremendous help in the past, especially in the conception of this thesis. Not forgetting Dan and my lab mates, past and present, without whom this learning experience would not have been complete. To my housemates, thank you Chi for the spontaneous fun; Bert for the walks and talks; Francis for his onomatopoeic care; Gavin for the good times running around the house; and Nathan for being there. I would also like to thank 'the kids'-Jeff, Paul, Daniel, Angela and Shirley for adding colors to life. Hanshien, Jerusha, Linda and Shipra, thank you for keeping me sane and helping me put things in perspective, despite being across time and space. A bulk of research done for this thesis would not have been possible without the generosity of the Rauch family and the College of Environment. Thank you for supporting me for the past two summers. Special thanks to Professor Elphick and Professor Hadler, who despite not being involved in the creation of this thesis have been a constant support to me throughout the years. I am indebted to the late Mr Houghton 'Buck' Freeman '43, whose radical generosity and passion continually inspires me. On multiple levels, this thesis would not have been at all possible without you. 'Thank you Mr. Freeman, your scholars proud we be.' Dad, Mum, Abigail and Aaron, thank you for your love, prayers and support. I would not have come this far without you. Above all I would like to thank the Creator for orchestrating every beautifully.

Lignin is an environmentally sustainable low‐impact starting material for making biofuels. The Taylor Lab at Wesleyan University investigates the pathways by which lignin is converted into central metabolites to help establish a path for the microbial conversion of lignin into a biofuel. LigAB, an extradiol dioxygenase from Sphingomonas paucimobilis SYK‐6, is one enzyme involved in this pathway, and is the focus of my research. The crystal structure of LigAB has revealed that phenylalanine‐103 (F103α) of LigAB is located proximal to the substrate‐binding site and is believed to contribute to the substrate specificity of the LigAB enzyme. F103α has been mutated to various smaller residues to test the hypothesis that it is functioning to limit the size of the substrate binding pocket, thus providing substrate specificity. The glutamic acid‐242 (E242β) residue, however, is essential to the coordination of the iron (II) ligand. Mutagenesis of E242β is anticipated to abolish iron binding, rendering the enzyme inactive. The histidine‐127 (H127β) and histidine‐195 (H195β) residues have been mutated to test their putative assignment as catalytic residues involved in acid‐base chemistry. Characterization of these mutants allows for better understanding of the substrate specificity and reaction mechanism of LigAB, which helps us learn about LigAB's function and about how to increase its promiscuity.