The catalytic activity of sulfatases is well understood, and it is known that their proper function depends intimately upon the presence of a formylglycine residue in the active site. Recent years have seen increased activity and interest in understanding the structure and function of the enzyme responsible for generating this formylglycine residue, Formylglycine Generating Enzyme (FGE). Originally thought to be a cofactorless enzyme, FGE catalyzes O<sub>2</sub>-dependent conversion of a cysteine to C(alpha)-formylglycine. Over the past few years it has become widely accepted that FGE is in fact a copper-dependent enzyme. However, despite these breakthroughs, thorough structural and electronic characterization of the copper active site has proven difficult. Here we have established a robust spectroscopic characterization of the copper active site and kinetic behavior of FGE in reaction with dioxygen. We have characterized the copper active site of resting FGE as well as the substrate bound form using x-ray techniques (x-ray absorption, x-ray emission, and extended x-ray absorption fine structure spectroscopies) as well as stopped-flow kinetic analyses. Stopped-flow kinetics combined with rapid freeze quench methods and low-temperature electron paramagnetic resonance spectroscopy have provided a window for us to study of the reaction cycle of FGE, and to provide evidence for several stages of the FGE reaction with a 14-mer peptide substrate and O<sub>2</sub>.

New insight into the reaction mechanism of the formylglycine generating enzyme: A spectroscopic perspective

254th National Meeting (2017)

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