Requirement for IscS in Biosynthesis of All Thionucleosides in Escherichia coli
AUTOR(ES)
Lauhon, Charles T.
FONTE
American Society for Microbiology
RESUMO
Escherichia coli tRNA contains four naturally occurring nucleosides modified with sulfur. Cysteine is the intracellular sulfur source for each of these modified bases. We previously found that the iscS gene, a member of the nifS cysteine desulfurase gene family, is required for 4-thiouridine biosynthesis in E. coli. Since IscS does not bind tRNA, its role is the mobilization and distribution of sulfur to enzymes that catalyze the sulfur insertion steps. In addition to iscS, E. coli contains two other nifS homologs, csdA and csdB, each of which has cysteine desulfurase activity and could potentially donate sulfur for thionucleoside biosynthesis. Double csdA csdB and iscS csdA mutants were prepared or obtained, and all mutants were analyzed for thionucleoside content. It was found that unfractionated tRNA isolated from the iscS mutant strain contained <5% of the level of sulfur found in the parent strain. High-pressure liquid chromatography analysis of tRNA nuclease digests from the mutant strain grown in the presence of [35S]cysteine showed that only a small fraction of 2-thiocytidine was present, while the other thionucleosides were absent when cells were isolated during log phase. As expected, digests from the iscS mutant strain contained 6-N-dimethylallyl adenosine (i6A) in place of 6-N-dimethylallyl-2-methylthioadenosine and 5-methylaminomethyl uridine (mnm5U) instead of 5-methylaminomethyl-2-thiouridine. Prolonged growth of the iscS and iscS csdA mutant strains revealed a gradual increase in levels of 2-thiocytidine and 6-N-dimethylallyl-2-methylthioadenosine with extended incubation (>24 h), while the thiouridines remained absent. This may be due to a residual level of Fe-S cluster biosynthesis in iscS deletion strains. An overall scheme for thionucleoside biosynthesis in E. coli is discussed.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135461Documentos Relacionados
- The Geobacillus stearothermophilus V iscS Gene, Encoding Cysteine Desulfurase, Confers Resistance to Potassium Tellurite in Escherichia coli K-12
- The Cysteine Desulfurase IscS Is Required for Synthesis of All Five Thiolated Nucleosides Present in tRNA from Salmonella enterica Serovar Typhimurium
- The iscS gene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H
- The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli
- Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS/IscU complex: Implications for the mechanism of iron-sulfur cluster assembly