Mechanistic and structural study of human aspartate/asparagine-beta-hydroxylase

The 2-oxoglutarate (2OG) dependent oxygenases are a superfamily of enzymes that utilise an Fe(II) cofactor and 2OG co-substrate for the catalysis of oxidative reactions. It is widely considered that an Fe(II)-binding motif comprising two histidine residues and a third acidic residue, thus creating an HXD/E...H triad, is a defining characteristic of 2OG oxygenase active sites. Aspartate/asparagine-β-hydroxylase (AspH) is a 2OG-dependent oxygenase that catalyses the hydroxylation of β-carbons of aspartate and asparagine residues in epidermal growth factor-like domains. By contrast with most other 2OG-dependent hydroxylases, AspH employs an Fe(II)-binding dyad, consisting of two histidine residues only. The significance of this unusual Fe(II)-binding motif and any effect this may have on the AspH mechanism is currently unknown. Chapter 1 provides an overview of the consensus mechanism for 2OG-oxygenases, and the current understanding of AspH structure and function. Chapter 2 describes studies investigating the nature of Fe(II)-binding at the AspH active site through the use of site-directed mutagenesis studies. The discovery that AspH retains significant levels of hydroxylation activity with a single active site Fe(II)-ligating histidine residue was investigated in detail. Chapter 3 describes work which explores the nature of 2OG-binding in AspH through the use of clinically relevant AspH variants. An unambiguous link between impaired AspH activity and pathogenic mutations in the AspH gene is established by characterisation of these AspH variants. Chapter 4 outlines work that establishes an AspH microcrystal system that could be used for future time-resolved serial X-ray crystallography experiments. A method for generating slurries of AspH microcrystals was developed and the system was tested at a synchrotron beamline. Additionally, a structure of AspH was obtained using serial crystallography at an X-ray free electron laser (XFEL) beamline. Attempts to recombinantly express a predicted 2OG-dependent oxygenase, Aspartate-β-hydroxylase domain-containing protein 1 (AspHD1), are outlined in Chapter 5. AspHD1 is predicted to possess a highly unusual RXG...H active site Fe(II) binding motif. Overall, as described in Chapter 6, this work characterises the unusual AspH Fe(II)-binding dyad and provides further insight into the mechanism of AspH catalysis. It is hypothesized that the side chains of AspH substrate aspartate residues may have a role in metal templating at the AspH active site. This hypothesis requires further consideration, but could have implications for the assignment of metal-ligating protein active sites