Crystal Structure of the Ergothioneine Sulfoxide Synthase from Candidatus Chloracidobacterium thermophilum and Structure-Guided Engineering To Modulate Its Substrate Selectivity

Abstract

Ergothioneine is a thiohistidine derivative with potential benefits on many aging-related diseases. The central step of aerobic ergothioneine biosynthesis is the oxidative C-S bond formation reaction catalyzed by mononuclear nonheme iron sulfoxide synthases (EgtB and Egt1). Thus far, only the Mycobacterium thermoresistibile EgtB (EgtB _Mth ) crystal structure is available, while the structural information for the more industrially attractive Egt1 enzyme is not. Herein, we reported the crystal structure of the ergothioneine sulfoxide synthase (EgtB _Cth ) from Candidatus Chloracidobacterium thermophilum. EgtB _Cth has both EgtB- and Egt1-type of activities. Guided by the structural information, we conducted Rosetta Enzyme Design calculations, and we biochemically demonstrated that EgtB _Cth can be engineered more toward Egt1-type of activity. This study provides information regarding the factors governing the substrate selectivity in Egt1- and EgtB-catalysis and lays the groundwork for future sulfoxide synthase engineering toward the development of an effective ergothioneine process through a synthetic biology approach.

Keywords: Rosetta Enzyme Design; enzyme engineering; ergothioneine; nonheme iron enzyme; sulfur-containing natural product.

Figure 1.

¹H NMR analysis of EgtB_Cth reactions. (A) EgtB_Cth reaction under EgtB reaction conditions. The two hydrogens of compound 2 imidazole side chain are labeled as 2, and the hydrogen of compound 3 imidazole hydrogen is labeled as 3. (B) EgtB_Cth reaction under Egt1-conditions. Compound 4 imidazole hydrogen is labeled 4. (C) Ratios of sulfoxide synthase and cysteine dioxygenase activity of EgtB_Cth under EgtB- and Egt1-type of reaction conditions. These two competing pathways are present in all three sulfoxide synthases (Egt1, EgtB, and OvoA). Notably, a significant level of cysteine dioxygenase activity was observed in EgtB_Cth-catalysis.

Figure 2.

Structures of EgtB_Cth and EgtB_Cth·hercynine binary complex. (A) Overall structure of EgtB_Cth in the tetrameric configuration with each monomer labeled. In Monomer A, the N-terminal domain (residue 17 to 183) is shown in blue and the C-terminal domain (residue 194 to 433) is shown in pink. The iron cofactor present at the active site of each monomer is shown as a brown sphere. (B) The 2mF_o-DF_c map of iron coordination site of EgtB_Cth contoured at 1.5σ (blue mesh); the metal ion is shown as a brown sphere and the coordinating residues are represented in sticks. Ordered water molecules coordinating the iron are shown as red spheres. (C) The mF_o-DF_c omit map of the active site of EgtB_Cth cocrystallized with hercynine contoured at 3σ (green mesh). The chemical structure of the substrate hercynine (shown as yellow sticks) was modeled into the positive density. (D) The interaction network between hercynine and EgtB_Cth active-site residues. Residues interacting with the substrate hercynine are shown in sticks with the potential interactions shown in black dash lines. (E) The previously reported structure of EgtB_Mth· dimethyl histidine·γ-Glu-Cys complex (PDB ID 4X8D) superimposed on the EgtB_Cth·herycine complex (shown in green). The side chains of the EgtB_Mth residues interacting with the γ-Glu-Cys are shown in sticks (white) and numbered with a superscript (′), the corresponding residues in the EgtB_Cth structure are shown as blue sticks. (F) The putative γ-Glu-Cys binding mode to EgtB_Cth (shown as yellow sticks). The potential interactions between γ-Glu-Cys and EgtB_Cth active-site residues were depicted as black dashed lines, and the side chains of the interacting residues are shown as blue sticks

Scheme 1.. Ergothioneine, Ovothiol Biosynthesis, and Distinct Properties of Sulfoxide Synthase EgtB_Cth^a

^a(A) Two aerobic ergothioneine biosynthetic pathways. (B) Ovothiol biosynthetic pathway. (C) Sequence similarity network analysis of ergothioneine sulfoxide synthases and the link between the sulfoxide synthase EgtB_Cth (in red), Egt1 (in blue), and EgtB (in green). (D) EgtB_Cth exhibits Egt1- and EgtB-type activities.