Polyuronic acids are an important constituent of seaweed and plants, and therefore represent a significant part of global biomass, providing an abundant carbon source for both terrestrial and marine heterotrophic bacteria. Through the action of polysaccharide lyases, polyuronic acids are degraded into unsaturated monouronic acid units, which are fed into the Entner-Doudoroff pathway where they are converted into pyruvate and glyceraldehyde-3-phosphate. The first step of this pathway was thought to occur non- enzymatically. A highly conserved sequence, kdgF was found in alginate and pectin utilization loci in a diverse range of prokaryotes, in proximity to well established enzymes catalyzing steps downstream in the Entner-Doudoroff pathway and I hypothesized that KdgF was involved in the catalysis of the first step of this pathway. The kdgF genes from both Yersinia enterocolitica and a locally acquired Halomonas sp. were expressed in Escherichia coli and their activity was examined using unsaturated galacturonic acid depletion activity assays. To gain perspective on the general structure of KdgF, x-ray crystallography was used to obtain a crystal structure of both HaKdgF and YeKdgF. These crystal structures provided insight into the molecular details of catalysis by the KdgF proteins, including their putative catalytic residues and a coordinated metal binding site for substrate recognition. To elucidate amino acids that may be involved in binding and/or catalysis, mutants were created in HaKdgF, and lack of activity was observed in four mutants (Asp102A, Phe104A, Arg108A, and Gln55A). The research done in this study suggests that KdgF proteins use a metal binding site coordinated by three histidines and several additional residues to cause a change in monouronic acid, thereby, affecting the unsaturated double bond. This suggests that KdgF is involved in the first step in the Entner-Doudoroff pathway, which is the linearization of unsaturated monouronic acids. / Graduate

Metal ions play essential roles in biological functions including catalysis, protein stability, DNA-protein interactions and cell signaling. It is estimated that 30% of proteins utilize metals in some fashion. Additionally, methods by which metal ions can be visualized have been utilized to study metal concentrations and localizations in relation to disease. Understanding the roles metals play in biological systems has great potential in medicine and technology. Chapters 1 and 2 of this dissertation analyzes the structure and function of the Mn-dependent enzyme oxalate decarboxylase (OxDc) and Chapter 2 presents a bioinformatic analysis of the cupin superfamily that provides the structural scaffold of the decarboxylase. The X-ray crystal structure of the W132F variant was determined and utilized together with EPR data to develop a computational approach to determining EPR spectra of the enzyme’s two metal-binding centers. Furthermore, a variant in which the catalytic Glu162 was deleted revealed the binding mode of oxalate, the first substrate-bound structure of OxDc. OxDc is a member of the cupin superfamily, which comprises a wide variety of proteins and enzymes with great sequence and functional diversity. A bioinformatics analysis of the superfamily was performed to analyze how sequence variation determines function and metal utilization. Chapters 3 and 4 discuss the expansion of lanthanide-binding tags (LBTs) to in cellulo studies. Lanthanide-binding tags are short sequences of amino acids that have high affinity and selectivity for lanthanide ions. An EGF-LBT construct used to quantify EGF receptors on the surface of A431 and HeLa cells. The results from the LBT quantification are consistent with previous studies of EGFR receptors in these cell types, validating the use of this method for future studies. The potential of using LBTs for X-ray fluorescence microscopy (XFM) was also investigated. LBT-labeled constructs were utilized to investigate if membrane bound as well as cytosolic LBT-containing proteins could be visualized and localized to their cell compartments via XFM; both membrane-localized and cytosolic proteins were successfully visualized. With the high resolution (< 150 Å) obtainable with new synchrotron beamline configurations LBTs could be used to study nanoscale biological structures in their near-native state.