Date of Award

January 2015

Degree Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Nigel G. J. Richards

Second Advisor

Lei Li

Committee Member 1

Kavita Shah

Committee Member 2

Andrew Mesecar


Oxalate decarboxylase (EC 4. 1. 1. 2 OxDC) from Bacillus subtilis is a manganese-dependent enzyme that catalyzes the cleavage of the chemically inactive C-C bond in oxalate to yield formate and carbon dioxide. A mechanism involving Mn(III) has been proposed for OxDC, however no clear spectroscopic evidence to support this mechanism has yet been obtained. In addition, a recent study has shown that N-terminal metal binding site loop variants of OxDC were able to catalyze the oxidation of oxalate to yield hydrogen peroxide and carbon dioxide, which makes OxDc function as another oxalate degradation protein in the cupin superfamily, oxalate oxidase (EC OxOx). In this work, wild-type (WT) OxDC and a series of variants with mutations on conserved residues were characterized to further investigate the catalytic mechanism of OxDC. The application of membrane inlet mass spectrometry (MIMS), electronic paramagnetic resonance (EPR) spectroscopy and kinetic isotope effects (KIEs) provided more detailed information about the mechanism. Mn(III) was identified and characterized under acidic conditions in the presence of dioxygen. Also, mutations on the second shell residues in the N-terminal metal binding site affected the properties of the metal. In the N-terminal domain, the functional importance of the residues in the active site loop region, especially Glu162, was confirmed, and evidence for the proposed mechanism in which OxDC and OxOx share the initial steps has been found. In addition, the mono-dentate coordination of oxalate in the N-terminal metal binding site was confirmed by X-ray crystallography. A proteinase cleavable OxDC was constructed and characterized, revealing a strong interaction between the N-terminal and C-terminal domains.