Browsing by Author "Carlson, Paul"

Efficient utilization of renewable plant and crop based biomass is one of the main areas of study in industrial biotechnology. Roughly 20-30 % of all plant biomass consists of hemicellulose, a polymer composed of six carbon (hexose) and five carbon (pentose) sugars. This proves a challenge in utilizing all available plant biomass efficiently, since many micro-organisms, which for instance readily ferment glucose (a hexose) into ethanol, will not do so for pentose sugars. Alternative pathways for utilization of pentose sugars are being looked for. Characterization of the Caulobacter crescentus D-xylonate dehydratase (Cc XyDHT) was the aim of this study. Cc XyDHT belongs to the ILVD/EDD protein family, whose members incorporate an iron-sulfur (FeS) cluster into their protein structure coordinated by two to four cysteine amino acids. Removal of the cysteine ligands should disrupt the incorporation of the FeS cluster, and thus presumably the activity of the enzyme. Three cysteines in Cc XyDHT which could act as ligands for the FeS cluster had previously been identified by sequence alignment among the ILVD/EDD family. Accordingly, four single cysteine-to-serine mutants of the Cc XyDHT as expression plasmid constructs were designed, with a non-conserved cysteine residue selected as a control. The wild type and mutated Cc XyDHTs were produced in Escherichia coli and purified with affinity chromatography using Strep-tag. SDS-page and Western blotting with an anti-Strep-tag antibody were used to confirm that the expressed proteins were Cc XyDHTs. The mutations’ effects on the protein fold and to the presence of the FeS cluster were investigated with UV and circular dichroism spectroscopy. Cc XyDHT catalyzes the conversion of D-xylonate into 2-keto-3-deoxy-xylonate. A colorimetric assay using thiobarbituric acid (TBA) as the reactant was used to measure enzymatic activity. The pH and temperature optima, substrate specificity, and enzyme kinetics of the wild type Cc XyDHT were determined. For the two best substrates, kcat/Km = 1220 mM/min for D-xylonate and kcat/Km = 1160 mM/min for D-gluconate were found. All three conserved mutations were found to reduce enzymatic activity more than 99 % with these substrates. Depending on which cysteine was disrupted, differences in the kinetic constants between D-xylonate and D-gluconate were found. The non-conserved mutation reduced activity by approx. 40 %. The spectroscopic results indicate that the three conserved mutants lacked the FeS cluster, while the non-conserved mutant still incorporated it. It can be concluded that the three conserved cysteines are involved in coordinating the FeS cluster, which itself plays an important role in the catalytic activity of Cc XyDHT. These findings should be helpful for follow-up studies in biotechnological application of pentose sugars acids and their derivatives.