Formation of Thiolated Nucleosides Present in tRNA from
            <i>Salmonella enterica</i>
            serovar Typhimurium Occurs in Two Principally Distinct Pathways

Leipuviene, Ramune; Qian, Qiang; Björk, Glenn R.

doi:10.1128/jb.186.3.758-766.2004

Cited by 46 publications

(56 citation statements)

References 70 publications

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“…(1 M) in the absence or presence of IscU (18.5 M), separated on spin column, and subjected to non-reducing SDS-PAGE. 35 S label incorporation was analyzed with a PhosphorImager. Protein migration positions determined by Coomassie Blue staining are indicated on the left.…”

Section: Discussionmentioning

confidence: 99%

“…Each of the other active site loop mutant proteins exhibited cysteine desulfurase activity that was equal to or greater than that of the wild-type enzyme. Furthermore, an in vitro assay of s 4 U biosynthesis using 35 S-labeled cysteine showed that each mutant protein (Ն1 M) was able to transfer the sulfur of cysteine to the appropriate tRNA substrate with similar efficiency, although the S326A mutant was consistently ϳ30 -40% lower at all of the concentrations tested (0.2-1 M). All of the mutant proteins showed s 2 U synthesis activity that was not significantly different from wild type at all of the concentrations tested.…”

Section: Iscs (L333a and S326a) Mutant Phenotypes Are Similar To Thosmentioning

confidence: 99%

“…The gene products known to be involved are indicated near the relevant step. The synthesis of a fourth thionucleoside, s 2 C, is largely uncharacterized but is known to require iscS and ttcA (35).…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…2). Jager et al (35) report that the ttcA gene required for s 2 C synthesis contains two required Cys-X-X-Cys motifs (35) that may be indicative of an Fe-S cluster protein. Recently, Leipuviene et al (36) reported additional evidence consistent with our results that supports the dual role of IscS in thionucleoside biosynthesis as depicted in Fig.…”

mentioning

confidence: 99%

“…Samples were immediately analyzed by gel electrophoresis using a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate in the absence of added reducing agent. 35 S was visualized by exposure on a phosphorimaging screen and analyzed using a Personal Molecular Imager FX (Bio-Rad) and analyzed with Quantity One quantitation software supplied with the instrument.…”

mentioning

confidence: 99%

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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

Journal of Biological Chemistry

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Iscs (L333a and S326a) Mutant Phenotypes Are Similar To Thosmentioning

confidence: 99%

Section: Enzyme Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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

Journal of Biological Chemistry

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Sulfur Modifications in tRNA: Function and Implications for Human Disease

Shigi

2016

Modified Nucleic Acids in Biology and Medicine

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Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

Sigel

Skilandat

Sigel

et al. 2012

Cadmium: From Toxicity to Essentiality

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Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromaticnitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono-to the di-and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono-or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand contains an aromatic residue (e.g., 2,2'-bipyridine or the indole ring of tryptophanate) intramolecular stack formation takes place. With buffers like Tris or Bistris mixed ligand complexes are formed. Cd(2+) coordination to dinucleotides and to dinucleoside monophosphates provides some insights regarding the interaction between Cd(2+) and nucleic acids. Cd(2+) binding to oligonucleotides follows the principles of coordination to its units. The available crystal studies reveal that N7 of purines is the prominent binding site followed by phosphate oxygens and other heteroatoms in nucleic acids. Due to its high thiophilicity, Cd(2+) is regularly used in so-called thiorescue experiments, which lead to the identification of a direct involvement of divalent metal ions in ribozyme catalysis.

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Formation of Thiolated Nucleosides Present in tRNA from Salmonella enterica serovar Typhimurium Occurs in Two Principally Distinct Pathways

Cited by 46 publications

References 70 publications

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

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

Sulfur Modifications in tRNA: Function and Implications for Human Disease

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

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