Project Title: Mechanistic characterisation of a cofactor-independent HPP oxygenase

Supervisor: Prof. Gerrit Poelarends, University of Groningen, The Netherlands

Objectives: The aim of the work is to elucidate the catalytic mechanism of a cofactor-independent monooxygenase that belongs to the tautomerase superfamily and catalyzes the oxidative cleavage of 4-hydroxyphenylenolpyruvate (HPP); see Baas et al., 2015, Biochemistry 54:1219-1232. The objectives are (1) to synthesize and test various HPP analogues as potential substrates or inhibitors of HPP oxygenase; (2) to determine X-ray crystal structures of HPP oxygenase in complex with substrate HPP and/or HPP analogues; (3) to perform mutagenesis studies to confirm the catalytic roles of identified active site residues.

Methodology: Chemical synthesis of HPP analogues (in collaboration with Syncom BV), enzyme assays and product analyses by various methods including LC-MS and UV and NMR spectroscopy (in collaboration with Prof. Susanne Fetzner), protein purification, crystallization and structure determination (in collaboration with Dr. Andy-Mark Thunnissen), structure-based site-directed mutagenesis, and steady-state kinetic analysis.

Expected Results: (i) Insight into the substrate scope of HPP oxygenase; (ii) Detailed understanding of the catalytic mechanism of HPP oxygenase.