Authors:

Hemanthie Wickramasinghe Thanthirige

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The formylglycine generating enzyme (FGE) converts a selected cysteine residue to a formylglycine residue in prokaryotes and eukaryotes. The FGE is a mononuclear copper containing enzyme and identified as an unusual oxidation catalyst due to the differences in the active site residues and active site geometry, which can differentiate FGE from other copper containing monooxygenases. The active catalytic site of FGE contains two cysteines from the FGE protein and one cysteine residue from the substrate. The objective of this study is to introduce selenocysteine in place of the substrate cysteine and extract catalytic active site information through X-ray absorption spectroscopy (XAS) techniques. The sulfur in cysteine and selenocysteine share similar chemical properties, therefore, replacing cysteine with selenocysteine is a possibility. But cysteine and selenocysteines have their own chemical differences, such as the pKa values. Since selenium is not natively present in FGE, but multiple cysteines are present, selenocysteine on substrate can be used as a probe in XAS experiments. The FGE protein from Thermomonospora curvata is produced through recombinant protein expression using Escherichia coli, purified by affinity chromatography, and followed by copper incorporation. FGE-27 is a substrate peptide which contains one cysteine at the sixth position and has been reported previously to bind to FGE. Currently, we are replicating and optimizing the synthesis of FGE-27 substrate in our laboratory. The modification of FGE-27 substrate to incorporate selenocysteine will be conducted as the next step to analyze FGE: FGE27_C6U system through XAS.

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Auburn University / College of Sciences and Mathematics / 2025

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Co-authors:

Hemanthie Wickramasinghe Thanthirige