Biochemical and structural characterization of a novel enzyme involved in uronic acid metabolism

• Main Author: Lee, Seung Hyae
• Other Authors: Boraston, Alisdair Bennett
• Language: English; en
• Published: 2014
• Subjects: metabolism; cupin superfamily; bacteria; x-ray crystallography; yersinia enterocolitica; halomonas; alginate; pectin; entner doudoroff pathway
• Online Access: http://hdl.handle.net/1828/5813

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