Cysteine Sulfinate Desulfinase, a NIFS-like Protein ofEscherichia coli with Selenocysteine Lyase and Cysteine Desulfurase Activities

170

180

IscS, a cysteine desulfurase implicated in the repair of Fe-S clusters, was recently shown to act as a sulfurtransferase in the biosynthesis of 4-thiouridine (s 4 U) in tRNA (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568). In frame deletion of the iscS gene in Escherichia coli results in a mutant strain that lacks s 4 U in its tRNA. Assays of cell-free extracts isolated from the iscS ؊ strain confirm the complete loss of tRNA sulfurtransferase activity. In addition to lacking s 4 U, the iscS ؊ strain requires thiamin and nicotinic acid for growth in minimal media. The thiamin requirement can be relieved by the addition of the thiamin precursor 5-hydroxyethyl-4-methylthiazole, indicating that iscS is required specifically for thiazole biosynthesis. The growth rate of the iscS ؊ strain is half that of the parent strain in rich medium. When the iscS ؊ strain is switched from rich to minimal medium containing thiamin and nicotinate, growth is preceded by a considerable lag period relative to the parent strain. Addition of isoleucine results in a significant reduction in the duration of this lag phase. To examine the thiazole requirement, we have reconstituted the in vitro biosynthesis of ThiS thiocarboxylate, the ultimate sulfur donor in thiazole biosynthesis, and we show that IscS mobilizes sulfur for transfer to the C-terminal carboxylate of ThiS. ThiI, a known factor involved in both thiazole and s 4 U synthesis, stimulates this sulfur transfer step by 7-fold. Extracts from the iscS ؊ strain show significantly reduced activity in the in vitro synthesis of ThiS thiocarboxylate. Transformation of the iscS ؊ strain with an iscS expression plasmid complemented all of the observed phenotypic effects of the deletion mutant. Of the remaining two nifS-like genes in E. coli, neither can complement loss of iscS when each is overexpressed in the iscS ؊ strain. Thus, IscS plays a significant and specific role at the top of a potentially broad sulfur transfer cascade that is required for the biosynthesis of thiamin, NAD, Fe-S clusters, and thionucleosides.

Section: Discussionmentioning

confidence: 77%

“…coli contains three genes with homology to nifS. In addition to iscS, these are cysteine-sulfinate desulfinase (33) and csdB (34). The csdB gene is identical to sufS, which was found to be required for the stable maintenance of the [2Fe-2S]-containing Fhuf protein in E. coli (35).…”

Section: Discussionmentioning

confidence: 99%

The iscS Gene in Escherichia coli Is Required for the Biosynthesis of 4-Thiouridine, Thiamin, and NAD

Lauhon

Kambampati

2000

170

180

“…The highest Z scores were calculated to be 62.4 for human SCL (an as yet unpublished structure, PDB 2HDY), 49 (42). This result shows that the structure of SCL is quite similar to that of human SCL, and more similar to those of group I cysteine desulfurases (IscS and NifS-like protein) than those of group II cysteine desulfurases (SufS and CsdB) (35,43) (Fig. 3B).…”

Section: Esi-ms Analysis Of Enzymeboundmentioning

confidence: 75%

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase

Omi

Kurokawa

Mihara

et al. 2010

Self Cite

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. UVvisible 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 seleniumcontaining substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

“…E. coli contains iscS and two other paralogs, csdA and sufS (csdB). These enzymes can be placed into two groups, based on sequence of an active site loop that contains an essential cysteine and other criteria (13). By these criteria, E. coli IscS is a group I enzyme, whereas SufS and CSD are members of group II.…”

mentioning

confidence: 99%

Substitutions in an Active Site Loop of Escherichia coli IscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesis in Vivo

Lauhon

Skovran

Urbina

et al. 2004

IscS catalyzes the fragmentation of L-cysteine to Lalanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys 328 resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys 328 identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s 2 C and ms 2 i 6 A) yet showed wild type levels of Fe-S-independent thionucleosides (s 4 U and mnm 5 s 2 U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.