Tatsuo Kurihara

CYSTEINE SULFINATE DESULFINASE, A NIFS-LIKE PROTEIN OF ESCHERICHIA COLI WITH SELENOCYSTEINE LYASE AND CYSTEINE DESULFURASE ACTIVITIES : GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF A NOVEL PYRIDOXAL ENZYME

Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. TheEscherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (M r43,238) and contains pyridoxal 5′-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms froml-cysteine, l-cystine,l-selenocysteine, and l-selenocystine to produce l-alanine. Because l-cysteine sulfinic acid was desulfinated to form l-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714–4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

Escherichia coli NifS-like Proteins Provide Selenium in the Pathway for the Biosynthesis of Selenophosphate

Selenophosphate synthetase (SPS), theselD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K m value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se0-generating system consisting ofl-selenocysteine and the Azotobacter vinelandiiNifS protein can replace selenide for selenophosphate biosynthesisin vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se0 and S0, respectively. In the present study, we demonstrate the ability of each E. coliNifS-like protein to function as a selenium delivery protein for thein vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.