HPDL, a yet uncharacterised mitochondrial enzyme: from cloning to first biophysical characterisation

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a largely uncharacterised, nuclear-encoded mitochondrial enzyme, expressed mainly in the central nervous system. It is thought to localize in the mitochondrial intermembrane space and play a role in tyrosine catabolism, specifically involved in ubiquinone synthesis pathway. Despite its involvement in several paediatric neurological disorders and cancers, only 14 studies, primarily focused on clinical descriptions of novel HPDL-related mutations, are indexed in PubMed, highlighting the scarcity of biochemical research on this protein. The lack of structural and catalytic data limits progress in understanding disease mechanisms and developing therapies. This thesis is the starting point of a bigger project led by Professor Gabriele Giachin (University of Padova) to elucidate HPDL’s structure and function, facilitating drug discovery for HPDL-related disorders. Given the absence of established methods for recombinant HPDL production, this thesis aims to develop an optimised approach for cloning, expression, and purification. Initial efforts focused on cloning HPDL from human genome (provided by Istituto Oncologico Veneto) both as a propeptide and without the predicted mitochondrial targeting sequence, into various plasmids for bacterial expression. Only the pET-SUMO with HPDL propeptide sequence construct was selected for protein production, because of its high expression yield and solubility across various expression conditions. In contrast, the mature form exhibited very poor expression and a tendency to aggregate, suggesting that the predicted targeting peptide may have been incorrect. Large-scale expression and purification trials over several attempts, lead to the selection of the optimal production media, and the outlining of a multi-step purification strategy: affinity chromatography with adenosine triphosphate (ATP) wash, desalting for fusion tag cleavage, secondary affinity chromatography under low-salt conditions, and size exclusion chromatography. Strong interactions between HPDL and bacterial chaperon GroEL during expression contributed to low protein yields despite HPDL’s solubility and observed fold stability. This led to the hypothesis that HPDL may interact with human chaperone Hsp60, suggesting a role in HPDL mitochondrial sorting. Biophysical characterization through circular dichroism (CD) spectroscopy confirmed that recombinantly produced HPDL folds correctly and demonstrates high stability against thermal denaturation. Temperature-dependent CD analysis also suggested a potential propensity for amyloid-like aggregation, an intriguing observation given HPDL's link to neurological disorders. Additionally, small-angle X-ray scattering (SAXS) revealed that HPDL exists as a monomer, challenging previous assumptions of a homodimeric structure based on its similarity to the dimeric paralogue 4-hydroxyphenylpyruvate dioxygenase (HPPD).