Abstract
Thiol dioxygenases (TDOs) are non‑heme Fe(II)‑dependent enzymes that catalyze the O(2)-dependent oxidation of thiol substrates to their corresponding sulfinic acids. Six classes of TDOs have thus far been identified and two, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), are found in eukaryotes. All TDOs belong to the cupin superfamily of enzymes, which share a common β‑barrel fold and two cupin motifs: G(X)(5)HXH(X)(3-6)E(X)(6)G and G(X)(5-7)PXG(X)(2)H(X)(3)N. Crystal structures of TDOs revealed that these enzymes contain a relatively rare, neutral 3‑His iron‑binding facial triad. Despite this shared metal-binding site, TDOs vary greatly in their secondary coordination spheres. Site‑directed mutagenesis has been used extensively to explore the impact of changes in secondary sphere residues on substrate specificity and enzymatic efficiency. This chapter summarizes site-directed mutagenesis studies of eukaryotic TDOs, focusing on the tools and practicality of non‑standard amino acid incorporation.