Biochemical and structural characterization of a novel enzyme involved in uronic acid metabolism
Date
2014-12-23
Authors
Lee, Seung Hyae
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Abstract
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.
Description
Keywords
metabolism, cupin superfamily, bacteria, x-ray crystallography, yersinia enterocolitica, halomonas, alginate, pectin, entner doudoroff pathway