Research Focus Areas
Our research projects mainly consist of two research fields. The first research field comprises of the study of biotechnological enzymes nitrilases, amidases and nitrile hydratases. The second field comprises of the structural proteomics studies of large mycobacterial protein complexes such as those from Mycobacterium tuberculosis (Mtb), the causative agent of TB.
Study of biotechnological nitrilases, amidases and nitrile hydratases
Nitrilases convert nitriles to the corresponding carboxylic acids, amidases convert amides to the corresponding carboxylic acids and nitrile hydratases convert nitriles to the corresponding amides. Of considerable interest in biocatalysis are the cysteine dependent amidases and nitrilases. The active sites of these enzymes share common features and both classes of enzyme have attracted attention as biocatalysts because of their specificity, enantioselectivity and regioselectivity. Nitrilases are also good candidates for the bioremediation of environments contaminated by toxic nitriles. The full potential of these enzymes as biocatalysts can only be achieved if the physical basis of their mechanism and specificity is known. This is not the case at the present time. In particular the nitrilases form oligomeric spirals via interfaces that are close to the active site as well as remote interfaces. In addition to issues concerning the specificity the mechanism of both the amidases and the nitrilases remain incompletely characterized. The goals of our research are thus to determine the structure of a helical nitrilase at near atomic resolution. Our approach utilizes structural bioinformatics, biochemical and biophysical analysis, quantum mechanical molecular modelling, site directed mutagenesis, cross-linking, mass spectroscopy, X-ray crystallography and three-dimensional electron microscopy to probe the structure, mechanism and specificity of the nitrilases and related enzymes. It is our intention to develop a novel, experimentally validated model that will provide a sound basis for the design of these enzymes for the production of multiple synthetic products.
Structural Proteomics of Large Mycobacterial Complexes
Current strategies aimed at eliminating Mycobacterium tuberculosis (Mtb), the causative agent of TB, are hampered by fundamental knowledge gaps with regard to the structural biology of Mtb. Only ~5% of a total 4031 Mtb H37Rv ORFs have been solved to atomic resolution. Furthermore, there is a strong bias in these structures towards monomers and dimers, because these are more amenable to recombinant expression and crystallization. In reality, most proteins function within large complexes (e.g. Kühner et al., 2009), but to sample this structurally underrepresented portion of the proteome, we need a different strategy. We have recently begun using native protein (to eliminate biases in recombinant expression) and cryo-electron microscopy (to prevent biases in crystallizability) to achieve this. Our strategy has been to fractionate the proteome sufficiently to allow us to perform three-dimensional reconstruction and identify protein complexes using mass spectrometry and structural homology.