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.

Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.

Background: Acid α-glucosidase, an enzyme replacement therapy for Pompe disease, is poorly targeted to lysosomes when relying on phosphomannose residues.Results: Fusing IGF-II to acid α-glucosidase resulted in more efficient uptake and glycogen clearance from muscle of Pompe mice.Conclusion: Enhanced binding to the cation-independent mannose 6-phosphate receptor (CI-MPR) enabled improved glycogen clearance in Pompe mice.Significance: BMN 701 is now being tested for Pompe disease in human clinical studies.

We have improved the in vitro assay for 4-thiouridine (s(4)U) biosynthesis in Escherichia coli tRNA by substituting an unmodified tRNA transcript as substrate and including recombinant ThiI protein, a known factor required for s(4)U synthesis. Using this assay, we have purified an enzyme from wild-type E. coli that is able to provide sulfur for s(4)U synthesis in vitro. The purified protein has a molecular weight of 45 kDa and contains pyridoxal phosphate as a cofactor. This protein catalyzes sulfur transfer from cysteine to tRNA and is analogous to factor C previously reported (Lipsett, M. N. (1972) J. Biol. Chem. 247, 1458-1461). UV spectroscopy and HPLC analysis of thiolated tRNA and its digests confirm that the product of the in vitro reaction is s(4)U. N-Terminal sequence analysis of the purified protein identifies it as IscS, a recently characterized NifS-like cysteine desulfurase that mobilizes sulfur for the synthesis of [Fe-S] clusters. We have cloned and overexpressed iscS and show that the recombinant protein displayed tRNA sulfurtransferase activity equal to that of the native protein. We also show that, of the multiple proteins in E. coli with cysteine desulfurase activity as observed by native gel staining, only IscS is able to mobilize the sulfur for transfer to tRNA. Our identification of IscS as a tRNA sulfurtransferase provides support for this activity in vivo and further expands the role for NifS proteins as versatile sulfur-carrying enzymes.