Structural characterization of two bacterial ?-Haloacid Dehalogenases: DehIVa and DehI

[Truncated abstract] α-Haloacid (αHA) dehalogenases cleave the carbon-halogen bonds of low molecular weight organic acids substituted at the Cα position with one or more halogen atoms. The addition of covalently bound halogens in most cases increases the toxicity of a compound and often makes the compound harder to break down. Accordingly, through the extensive use of these compounds in agriculture and industry as pesticides, herbicides, organic solvents, and reaction intermediates, many αHAs have been globally classified as priority pollutants. Hazardous αHAs such as the herbicide Dalapon (2,2-dichloropropionic acid) are no longer in global production (and are banned in some countries) as they have been identified as contaminants in drinking water owing their relatively high water solubility and difficulty to contain. In light of this it should be noted that numerous bacterial strains able to degrade αHAs have been isolated and all have been found to express dehalogenase enzymes. Interest in αHA dehalogenases comes from two complimentary areas; 1) Application in bioremedial practices; the decontamination of sites polluted with αHAs. 2) Application in chemical industries; to make use of αHA dehalogenases in the manufacture of alternative compounds, replacing older and often less efficient non-biological technologies1. To gain a better understanding of the biochemical function of an enzyme, an in-depth biochemical analysis is of considerable value. However, once you have an idea of the reaction chemistry, of substrate specificities and the residues likely to be involved, a structural characterisation of the biomolecule is required to give insights to the reaction mechanism itself. ... The identification of a hydrolytic water located ideally located to cleave the intermediate ester bond to complete the dehalogenation reaction is a highlight of the model. pH and temperature reduction allowed the prevention of the final hydrolytic reaction and the trapping of the substrate intermediate. The group I αHA dehalogenase that was studied, DehI, was originally isolated from Pseudomonas putida strain PP3 grown on Dalapon. Thought to be an isolated group of enzymes without any known phylogenetic links to other protein families, the reported structure of DehI is the first group I αHA dehalogenase to be solved. A symmetrical duplication in the structure enabled the identification of a very interesting evolutionary connection to the functionally unrelated CMD (carboxymuconolactone decarboxylase) family of proteins. In addition the enzyme's active site was identified for the first time and a series of hypotheses were made for L- and D-enantiomer binding interactions. Known to involve a direct attack by the hydrolytic water on the substrate chiral centre, unique to all reported hydrolytic dehalogenases, a reaction mechanism was also proposed in the context of the DehI structure. A NOTE ON THESIS LAYOUT: This thesis is presented as a series of manuscripts either published or submitted for publication. Each manuscript forms a self-contained chapter with its own references. Formatting for each was modified to make it consistent across the whole thesis. Included in the thesis are a General Introduction and a General Discussion, linking the manuscripts together to present a uniform body of research.