LAUSR

ConspectusIn recent years, considerable progress has been made toward elucidating the geometric and electronic structures of thiol dioxygenases (TDOs). TDOs catalyze the conversion of substrates with a sulfhydryl group to their sulfinic acid derivatives via the addition of both oxygen atoms from molecular oxygen. All TDOs discovered to date belong to the family of cupin-type mononuclear nonheme Fe(II)-dependent metalloenzymes. While most members of this enzyme family bind the Fe cofactor by two histidines and one carboxylate side chain (2-His-1-carboxylate) to provide a monoanionic binding motif, TDOs feature a neutral three histidine (3-His) facial triad. In this Account, we present a bioinformatics analysis and multiple sequence alignment that highlight the significance of the secondary coordination sphere in tailoring the substrate specificity and reactivity among the different TDOs. These insights provide the framework within which important structural and functional features of the distinct TDOs are discussed.The best studied TDO is cysteine dioxygenase (CDO), which catalyzes the conversion of cysteine to cysteine sulfinic acid in both eukaryotes and prokaryotes. Crystal structures of resting and substrate-bound mammalian CDOs revealed two surprising structural motifs in the first- and second coordination spheres of the Fe center. The first is the presence of the abovementioned neutral 3-His facial triad that coordinates the Fe ion. The second is the existence of a covalent cross-link between the sulfur of Cys93 and an ortho carbon of Tyr157 (mouse CDO numbering scheme). While the exact role of this cross-link remains incompletely understood, various studies established that it is needed for proper substrate Cys positioning and gating solvent access to the active site. Intriguingly, bacterial CDOs lack the Cys-Tyr cross-link; yet, they are as active as cross-linked eukaryotic CDOs.The other known mammalian TDO is cysteamine dioxygenase (ADO). Initially, it was believed that ADO solely catalyzes the oxidation of cysteamine to hypotaurine. However, it has recently been shown that ADO additionally oxidizes N-terminal cysteine (Nt-Cys) peptides, which indicates that ADO may play a much more significant role in mammalian physiology than was originally anticipated. Though predicted on the basis of sequence alignment, site-directed mutagenesis, and spectroscopic studies, it was not until last year that two crystal structures, one of wild-type mouse ADO (solved by us) and the other of a variant of nickel-substituted human ADO, finally provided direct evidence that this enzyme also features a 3-His facial triad. These structures additionally revealed several features that are unique to ADO, including a putative cosubstrate O2 access tunnel that is lined by two Cys residues. Disulfide formation under conditions of high O2 levels may serve as a gating mechanism to prevent ADO from depleting organisms of Nt-Cys-containing molecules.The combination of kinetic and spectroscopic studies in conjunction with structural characterizations of TDOs has furthered our understanding of enzymatic sulfhydryl substrate regulation. In this article, we take advantage of the fact that the ADO X-ray crystal structures provided the final piece needed to compare and contrast key features of TDOs, an essential family of metalloenzymes found across all kingdoms of life.