Project Title: Discovery of new cofactor-independent oxygenases

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

Objectives: The aim of the work is to elucidate the physiological function and substrate range of novel cofactor-independent oxygenases that belong to the tautomerase superfamily. These oxygenases will be identified on the basis of sequence & structure similarity to a cofactor-independent 4-hydroxyphenylenolpyruvate (HPP) monooxygenase (see Baas et al., 2015, Biochemistry 54:1219-1232). The objectives are (1) to identify HPP oxygenase-like sequences by bioinformatics tools; (2) to express, purify and functionally characterise newly identified cofactor-independent oxygenases; (3) to determine the X-ray crystal structure of the most interesting enzyme.

Methodology: Bioinformatics (BLASTP, structure-based sequence aligments, genomic context analyses, etc.), biotransformations and product analyses by various methods, including LC-MS and UV and NMR spectroscopy (in collaboration with Prof. Susanne Fetzner and DSM Food specialties BV), protein expression, purification, crystallization, and structure determination by X-ray crystallography (in collaboration with Dr. Andy-Mark Thunnissen).

Expected Results: (i) New cofactor-independent oxygenases identified within the tautomerase superfamily; (ii) Insight into the physiological function of these cofactor-independent oxygenases.