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.
Highlights
The most common selenium source in normal foods is selenocysteine and selenomethionine in proteins [8]
selenocysteine lyase (SCL) is a pyridoxal 5Ј-phosphate (PLP) enzyme that catalyzes the removal of selenium from L-selenocysteine to yield L-alanine
UV-visible spectrum analysis shows that loss of Cys-375 resulted in the formation of a nonproductive adduct with selenocysteine, suggesting that Cys-375 serves as a guide to direct the substrate in an appropriate orientation for the catalytic reaction
Summary
Materials—L-Selenocystine and L-cysteine were purchased from Sigma Aldrich. Selenopropionate was synthesized as described in supplemental Experimental Procedures. Crystals of SCL1⁄7L-cysteine and SCL1⁄7selenopropionate were obtained at 293 K by soaking the native crystals in the reservoir solution adjusted to pH 8.0. These crystals are isomorphous with the orthorhombic space group P212121, average unit cell parameters a ϭ 55.0, b ϭ 101.9, c ϭ 197.3 Å. X-ray diffraction data sets for the unliganded SCL (pH 8.0) were collected to 2.0 Å resolution on the BL5A station at the Photon Factory, KEK (Tsukuba, Japan). X-ray diffraction data sets for the SCL1⁄7L-cysteine crystal and the SCL1⁄7selenopropionate crystal were collected to 1.85 and 1.55 Å resolution on the BL41XU and the BL38B2 station, respectively, at SPring (Hyogo, Japan).
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