Reaction mechanism and molecular basis for selenium/sulfur discrimination of selenocysteine lyase

Abstract

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5'-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

FIGURE 1.

Deconvoluted ESI-mass spectra of SCL. Wild-type SCL (A–C) or the C375A mutant enzyme (D–F) was incubated with l-cysteine (B, E), l-selenocysteine (C, F), or none (A, D) and analyzed by ESI-mass spectrometer as described under “Experimental Procedures.” The peak at the mass of 47,353 for the [M + 80] species that corresponds to the selenopersulfide form of SCL (SCL-S-Se⁻) is indicated by an asterisk.

FIGURE 2.

UV-visible spectroscopic analysis of SCL. A, absorption spectra of wild-type SCL incubated with various concentrations (0, 15, and 30 μm, indicated by lines 1, 2, and 3, respectively) of l-selenocysteine. B, absorption spectra of wild-type SCL incubated with various concentrations (0, 0.027, 0.1, 0.26, 0.6, 1.25, 2.5, 5, 10, 20, and 40 mm, indicated by lines 1–11, respectively) of l-cysteine. C, absorption spectra of the C375A mutant incubated with various concentration (0, 1.5, 15, and 30 μm, indicated by lines 1–4) of l-selenocysteine. D, absorption spectra of the C375A mutant incubated with various concentrations (0, 0.0024, 0.0073, 0.027, 0.1, 0.26, 0.6, 1.25, 2.5, 5, 10, 20, and 40 mm, indicated by lines 1–13) of l-cysteine. Absorption spectra of SCL were recorded with a Shimadzu UV-visible spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) at pH 7.0.

FIGURE 3.

Overall structure of SCL. A, a dimeric form viewed down the crystallographic 2-fold axis. The upper half of the molecules is the superimposition of one subunit in the liganded closed form onto that in the unliganded open form by least-squares fitting of Cα atoms in the large domain. The open form is pink, and the closed form is blue with a red extended lobe. The lower half of the molecule represents the other subunit in the closed form. The small domain, the large domain, and the N-terminal loop are drawn in gray, green, and orange, respectively. The coenzyme PLP and l-cysteine located in the domain interface are represented by CPK models. The extended lobe in the open form of SCL is disordered and, therefore, not shown in the model. B, superpositioning of Cα atoms of SCL onto those of cysteine desulfurases. SCL complexed with l-cysteine, human SCL, T. maritima NifS-like protein complexed with l-cysteine (PDB ID: 1ECX), and E. coli CsdB complexed with l-propargylglycine (1I29) are presented in red, orange, blue, and green, respectively. The extended lobes are shown in thick lines. The PLP moieties, bound ligands, and active site lysine and cysteine residues are represented by stick models. A part of the extended lobe of T. maritima NifS-like protein is disordered and not shown in the model.

FIGURE 4.

Active site structures. A, SCL complexed with l-cysteine and B, SCL complexed with selenopropionate. The PLP moiety, active site residues, and bound ligands (A, l-cysteine; B, selenopropionate) are shown as ball-and-stick models. Water molecules (W1-W5) are presented as red circles. Backbone structures of the large domain, the small domain, and the extended lobe in one subunit are drawn in green, gray, and red, respectively. Cα structures and residues (indicated by asterisks) in the other subunit of the dimer are shown in blue. The 2F_o − F_c electron density map contoured at 1.0 σ is calculated from the final model with the bound ligand omitted. Both conformers of selenopropionate are drawn in B.