Structures of SenB and SenA enzymes from Variovorax paradoxus provide insights into carbon-selenium bond formation in selenoneine biosynthesis

Abstract

Selenoneine, an ergothioneine analog, is important for antioxidation and detoxification. SenB and SenA are two crucial enzymes that form carbon-selenium bonds in the selenoneine biosynthetic pathway. To investigate their underlying catalytic mechanisms, we obtained complex structures of SenB with its substrate UDP-N-acetylglucosamine (UDP-GlcNAc) and SenA with N-α-trimethyl histidine (TMH). SenB adopts a type-B glycosyltransferase fold. Structural and functional analysis of the interaction network at the active center provide key information on substrate recognition and suggest a metal-ion-independent, inverting mechanism is utilized for SenB-mediated selenoglycoside formation. Moreover, the complex structure of SenA with TMH and enzymatic activity assays highlight vital residues that control substrate binding and specificity. Based on the conserved structure and substrate-binding pocket of the type I sulfoxide synthase EgtB in the ergothioneine biosynthetic pathway, a similar reaction mechanism was proposed for the formation of C-Se bonds by SenA. The structures provide knowledge on selenoneine synthesis and lay groundwork for further applications of this pathway.

Keywords: Carbon–selenium bond; Ergothioneine; Glycosyltransferase; Selenoneine; Selenoneine synthase; SenA; SenB.

Fig. 1

Key steps in the ergothioneine and selenoneine biosynthetic pathways. Top, C–S bond formation catalyzed by type-I EgtB in Mycobacterium smegmatis. Bottom, C–Se bond formation by SenB and SenA. The difference between ergothioneine and selenoneine is labeled in red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Structural analysis of the glycosyltransferase SenB in complex with UDP-GlcNAc. (A) Overall structure of the SenB-UDP-GlcNAc binary complex in two vertical views. SenB and the ligand UDP-GlcNAc are shown as cartoons and spheres, respectively. α-helices are colored marine, β-sheets light-orange, loops light-gray while linker between N- and C-terminus dark-gray. The N- and C-termini of SenB are labeled with black dots. (B) The mFo-DFc omit map for the ligand UDP-GlcNAc was constructed at σ-level = 2.0 (black mesh). (C) Electrostatic potential map showing the substrate-binding pocket. (D) The interaction network of the ligand UDP-GlcNAc with SenB. The hydrogen bonds formed for the stabilization of UDP-GlcNAc are shown as green dashed lines. The residue names for hydrogen bond formation and the distance between hydrogen bonds are shown in green and black, respectively. The unit of the hydrogen bond length is angstroms (Å). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Structural analysis of the selenoneine synthase SenA in complex with TMH. (A) Overall structure of the SenA-TMH complex. The N- and C-terminal domains of SenA are shown as pale-green cartoons and surfaces, the ligand TMH and two imidazoles are shown as yellow sticks, and nickel ions are shown as gray spheres. The N- and C-termini are marked with black dots. (B) The mFo-DFc omit map of the coordination site contoured at 2σ (gray mesh). Residues and ligands are shown in the same color as in panel (A). Inset, the other view showing the coordination sites. GOL indicates the glycerol observed in the structure. Hydrogen bonds are shown as yellow-orange dashed lines. (C) Structural alignment between SenA and MthEgtB. SenA and its ligands are shown as in panel (A), while MthEgtB and its substrates TMH and γ-Glu-Cys and manganese ions are shown as cyan cartoons, cyan sticks and purple spheres, respectively. The substrate-binding pocket is shown in the yellow-orange dashed box and is enlarged for analysis in panel (D). (D) Comparison of the substrate binding pockets between SenA and MthEgtB. Residues and ligands of MthEgtB and SenA are shown in the same colors as those in panel (C). The dashed orange oval indicates the possible binding volume for the selenosugar substrate SenA. Water molecules are shown as red spheres and labeled with the letter W. (E) Relative catalytic activities of the SenA mutants. The data were quantified from three replicates with average and SD shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)