Biochemical discrimination between selenium and sulfur 1: a single residue provides selenium specificity to human selenocysteine lyase

Abstract

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

Figure 1. Structure of Human SCL.

A) Human SCL homodimer in complex with the co-factor PLP. The C388 residues of both subunits are indicated. B) Superposed subunits A and B of human SCL showing the structural differences in the active site segment and positioning of C388 (subunit A: pink “closed” and subunit B: cyan “open”), the location of Asp 146 is also shown.

Figure 2. Electron density for the dynamic segment.

A and B) Stereo figure of an Fo-Fc difference electron density omit map of the segment surrounding C388 for chains A and B, contoured at 2.5σ (0.11 eÅ-3).

Figure 3. Active site.

A) Human SCL in pink showing the PLP binding site and E. coli NifS/CsdB (PDB: 1KMK) with the Cys-sulfoselenide intermediate in blue showing the similar positioning of the C388-sulfur atom (hSCL) and that of C364 in E. coli NifS/CsdB. A second Sec substrate molecule, bound to the PLP in the E. coli NifS/CsdB , is also shown. B) Electron density for the C388 persulfide in subunit A after a 2 h Cys soak of a P1 spacegroup hSCL crystal. Fo-Fc difference electron density map of the C388 persulfide with the δ-sulfur omitted contoured at 4σ (0.23 eÅ-3). C) Position of mutated residues D146K, V256S and H389T in relation to C388 and the PLP cofactor.

Figure 4. Alignment of representative sequences of bacterial SCL/CD enzymes (Synechocystis SufS, E. coli NifS, E. coli IscS and T. maritima NifS) and Mammalian Sec-specific SCLs (Mouse, Rat and Human).

Residue positions corresponding to D146, V256 and H389 (hSCL numbering) are indicated with a yellow, green and purple background respectively.

Figure 5. Activity of wild-type and variant proteins.

A) Activity of WT hSCL and variants containing the D146K substitution with Cys. WT hSCL, solid black line and open circles; D146K/V256S/H389T, dotted grey line and closed diamonds; D146K, dot-dash black line and stars; D146K/V256S, dash black line and open squares; D146K/H389T, solid black line and filled triangles. B) Activity of WT hSCL and the D146K/H389T variant with Sec. WT, black line and open circles; D146K/H389T variant, grey line and open squares.

Figure 6. Conformations of the active site dynamic segment (blue).

A) Closed, B) Open. The sulfur atom of C388 is shown in yellow.

Figure 7. Potential mechanism of selenide product delivery directly to selenophosphate synthetase by the C388-containing flexible active site segment.