Differences in the Second Coordination Sphere Tailor the Substrate Specificity and Reactivity of Thiol Dioxygenases

Abstract

In 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.

Figure 1.

TDO SSNs. (A) The final TDO network (E-value of 1×10⁻³⁴) contains several large clusters. Importantly, unlike CDOs and MDOs, ADOs and CDOs exhibit independent clustering indicative of disparate biological functions. (B) Taxonomic filtering of the SSN reveals clustering coincident with separate organism domains and clades. The SAR clade includes stramenopiles, alveolates, and Rhizaria. Unique grouping of the viridiplantae clade on the cluster of Swiss-Prot reviewed PCOs marks a departure from the opisthokonta designation observed with Swiss-Prot reviewed eukaryotic CDOs.

Figure 2.

Sequence alignment showing conserved residues within TDOs. The sequences of the PCO4 from Arabidopsis thaliana (AtPCO4), the MDO from Pseudomonas aeruginosa (PaMDO), and the CDOs from Mus musculus (MmCDO) and Bacillus subtilis (BsCDO) are compared to the Mus musculus ADO (MmADO) sequence. Important ADO and PCO residues are highlighted in green, MDO in purple, CDO in blue, and key differences in yellow. All proteins possess a 3-His metal-binding motif marked by a ♦ and highlighted in gray. Additional motifs discussed in the text are denoted as follows: # Phe89 (MmADO numbering), ■ Cys-Tyr crosslink in MmCDO, ■ putative ADO and PCO crosslink motifs, • Cys120 and Cys169 in MmADO are replaced by Ser118 and Thr153 in PCO4, ▲ Asp192, * denotes a cis-peptide bond, and ♠ Tyr198 in MmADO and Tyr182 in AtPCO4 adjacent to the cis-peptide bond.

Figure 3.

Active site region of Cys-bound CDO (PDB code 4JTO). The Fe ion (orange sphere) is coordinated by three His residues (lavender) and substrate Cys (maroon). Other important active site residues are highlighted in dark purple. Hydrogen bonding interactions between amino acid residues and with substrate Cys are represented by dashed and dotted lines, respectively. The * marks the two consecutive cis-peptide bonds that help position Tyr 157.

Figure 4.

Active site region of 3-hydroxypropionic acid (3-HPA)-bound AvMDO (PDB code 6XB9). The Fe ion (orange sphere) is bound by three His residues (lavender) and the substrate analogue 3-HPA (blue). Other significant active site amino acids are highlighted in teal. Hydrogen bonding interactions between amino acid residues and with 3-HPA are represented by dashed and dotted lines, respectively.

Figure 5.

Active site region of AtPCO4 (PDB code 6S7E). The Fe ion (orange sphere) is bound by three His residues (lavender). Other important active site amino acids are highlighted in red. The arrow points to the hairpin loop and the * marks the cis-peptide bond adjacent to Tyr182.

Figure 6.

(A) MCD spectra at 4.5 K of Cys-bound CDO (top) and 2-AET-bound ADO (bottom). The distinct regions of the S(Cys) → Fe(II) and S(Cys) → Fe(III) charge transfer transitions are indicated. (B) X-band EPR spectra at 20 K of cyanide/Cys-bound Fe(III)CDO (top), 2-AET/cyanide-bound Fe(III)ADO (middle), and 2-AET/cyanide-bound Tyr208Phe Fe(III)ADO (bottom).

Figure 7.

Active site region of MmADO (PDB code 7LVZ). The Fe ion (orange sphere) is bound by three His residues (lavender). Other key amino acids in the active site are highlighted in red. The arrow points to the hairpin loop and the * marks the cis-peptide bond that aids in the positioning of Tyr198.

Scheme 1.

Oxidation of thiol-containing substrates catalyzed by TDOs.

Scheme 2.

Oxidation of Cys to CSA catalyzed by CDO.

Scheme 3.

Formal electronic configurations of the proposed lowest energy reaction intermediates for the oxidation of Cys by Fe(II)CDO.

Scheme 4.

Oxidation of 3-MPA to 3-sulfinopropionic acid catalyzed by MDO.

Scheme 5.

Oxidation of Nt-Cys peptides to the corresponding sulfinic acids catalyzed by PCO.

Scheme 6.

Reactions catalyzed by ADO. A. Oxidation of 2-AET to hypotaurine. B. Oxidation of Nt-Cys peptides to their corresponding sulfinic acids.