PhD defence H. Poddar

Structural and functional characterization of tautomerase and aspartase/fumarase superfamily enzymes

The thesis of Harshwardhan Poddar describes the structural, functional and mechanistic characterization of three enzymes belonging to the tautomerase and aspartase/fumarase superfamily of enzymes.

Biocatalysis is a branch of biochemistry that exploits the ability of an enzyme to convert a substrate into a compound of economic value in an environmentally friendly manner. Enzymes are known to catalyze complex chemical reactions in their active site pockets and it is important to understand the intricacies of the reaction mechanism at the molecular level. X-ray crystallography, a powerful technique, is used to calculate the position of individual atoms comprising an enzyme in the three-dimensional space. Solving structures of enzymes in complex with a ligand tells us how a compound binds in the enzyme active site and the role of the different amino acid residues during catalysis. This knowledge further enables the engineering of enzymes for efficient and selective catalysis.

4-Oxalocrotonate tautomerase (4-OT) is a versatile enzyme capable of promiscuously catalyzing synthetically useful C-C bond-forming reactions. The mechanism of these important reactions has been elucidated by solving structures of the wild-type and engineered 4-OT variants in complex with substrates. In addition, a new member of the tautomerase superfamily, RhCC, was identified and shown to catalyze a cofactor-independent oxygenation reaction, previously unseen in any other member of this superfamily. Finally, a new enzyme capable of catalyzing C-N bond forming reactions, called ethylenediamine-N,N’-disuccinic acid lyase (EDDS lyase), was identified in the aspartase/fumarase superfamily of enzymes. Crystal structures in complex with substrates and product gave important insights into the mechanism and synthetic potential of this robust enzyme. These three enzymes have shown their potential in the production of valuable compounds such as precursors for pharmaceuticals and various food additives, and the work described in this thesis brings forth new opportunities for further research into the optimization of the properties of these enzymes for industrial applications.

Promotores: Prof.dr. G.J. Poelarends and Prof.dr. W.J. Quax