Crystal structures reveal how distinct sites on the cysteine desulfurase IscS bind two different sulfur-acceptor proteins, IscU and TusA, to transfer sulfur atoms for iron-sulfur cluster biosynthesis and tRNA thiolation.

The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE•GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE•GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.

In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an α2β2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent.

(2014) Modification of the wobble uridine in bacterial and mitochondrial tRNAs reading NNA/NNG triplets of 2-codon boxes, RNA Biology, 11:12, 1495-1507

Cell wall extracts from the double-mutant mnn1 mnn9 strain were used as the immunogen to obtain a monoclonal antibody (MAb), SAC A6, that recognizes a specific mannoprotein-which we have named Icwp-in the walls of cells of Saccharomyces cerevisiae. Icwp runs as a polydisperse band of over 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Zymolyase extracts of cell walls, although an analysis of the secretory pattern of the mannoprotein shows that at the level of secretory vesicles, it behaves like a discrete band of 140 kDa. Immunofluorescence analysis with the MAb showed that Icwp lies at the inner layer of the cell wall, being accessible to the antibody only after the outer layer of mannoproteins is disturbed by treatment with tunicamycin. The screening of a gt11 expression library enabled us to identify the open reading frame (ORF) coding for Icwp. ICWP (EMBL accession number YLR391w, frame ؉3) codes for 238 amino acids, of which over 40% are serine or threonine, and contains a putative N-glycosylation site and a putative glycosylphosphatidylinositol attachment signal. Both disruption and overexpression of the ORF caused increased sensitivities to calcofluor white and Congo red, while the disruption caused an increased sensitivity to Zymolyase digestion, suggesting for Icwp a structural role in association with glucan.The cell wall of Saccharomyces cerevisiae is made up of three components, namely, glucans, mannoproteins, and chitin, and represents some 20% of the dry weight of the cell. It consists of a layered structure, with an internal layer made up of ␤-1,3 and ␤-1,6 glucans, small amounts of chitin and mannoproteins, and an outer layer of mannoproteins (13, 23). The inner layer is responsible for the shape and mechanical strength of the wall (19,24,50), while the outer mannoprotein layer determines the surface properties of the cell, such as hydrophobicity, electrical charge, flocculence, and sexual agglutinability, as well as limiting the porosity of the cell wall (8-10, 50).The mannoproteins can be divided into three groups according to the methods used for their extraction from the cell wall: sodium dodecyl sulfate (SDS)-extractable mannoproteins (44), glucanase-extractable mannoproteins, which can be released only after glucanase digestion of the glucan layer (31,44,47), and mannoproteins extractable by reducing agents (35). The glucanase-extractable mannoproteins identified so far have two common characteristics: one is a high serine/threonine content (up to 50% of the C-terminal half of the protein), and the other is the presence of a putative glycosylphosphatidylinositol (GPI) attachment site (23,46). Several of the proteins characterized so far, such as the sexual ␣-agglutinin (27, 29, 30), the anchor subunit of the a-agglutinin (38), and the flocculin encoded by the FLO1 gene, play a role on the surface of the cell (42). In these proteins, the highly O-glycosylated Cterminal half may endow them with a rod-like structure (22) that facilitates the exposure of their ...

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In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In and , however, cmnm5 appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm5) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm5 into nm5, whereas MnmC(m) converts nm5 into mnm5, thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that and are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm5s2U by MnmEG-MnmC in vivo avoids build-up of intermediates in . We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.tRNA contains modified nucleosides that are posttranscriptionally generated by the activity of specific enzymes (http: //modomics.genesilico.pl/sequence?seqtypeϭtRNA; 2). Many of these modifications frequently appear in the anticodon wobble position and are pivotal in the decoding process by stabilizing correct codon-anticodon interactions (1, 9). In Escherichia coli, the enzymes MnmE and MnmG carry out the GTPand flavin adenine dinucleotide (FAD)-dependent incorporation of the cmnm (CH 2 -NH-CH 2 -COOH) group at position 5 of the wobble uridine in several tRNAs, a reaction whose specific steps remain to be elucidated (4,7,8). MnmG and MnmE form a heterotetrameric ␣ 2 ␤ 2 complex in vitro (8). MnmG is a highly conserved FAD-binding protein (8) with 49% sequence identity to human MTO1.We cloned E. coli MnmG (MnmG Ec ; NCBI gi:2367273), MnmG , and MnmE (gi:12518545) into a modified pET15b vector (Novagen). The proteins were expressed in E. coli BL21(DE3) (Novagen). Cells were grown in LB medium, induced with 100 M isopropyl-1-thio-␤-D-galactopyranoside, and incubated for ϳ16 h at 16°C. Proteins were purified using nickelnitrilotriacetic acid (Ni-NTA) resin (Qiagen, Mississauga, Ontario, Canada) and concentrated to ϳ8 mg/ml in 20 mM Tris-HCl (pH 8.0), 0.8 M NaCl, 5% (vol/vol) glycerol, 5 mM dithiothreitol (DTT). MnmG Ec was crystallized by the hanging-drop method, equilibrating the protein drop against reservoir solution containing 100 mM Tris-HCl (pH 7.5), 100 mM sodium formate, 6.5% (wt/vol) polyethylene glycolPEG 8000, 6% (vol/vol) ethylene glycol. The crystals belong to space group P2 1 , with a ϭ 85.9 Å, b ϭ 144.1 Å, c ϭ 147.6 Å, and ␤ ϭ 106.8°, with four molecules in the asymmetric unit (V m ϭ 3.03 Å 3 Da Ϫ1 ) (6). They differ from the previously reported crystals and diffract to significantly higher resolution (7). Selenomethionine-labeled protein [expressed in strain DL41(DE3)] crystallized under the same conditions. Crystals of MnmG were obtained at 20°C by equilibrating protein (7 mg/ml) supplemented with the anticodon stem-loop fragment of tRNA Glu (Dharmacon) (molar ratio, 1:1.2) against a reservoir solution of 1.3 M Li 2 SO4, 0.1 M Tris-HCl (pH 8.0). They belong to space group P3 1 21, with a ϭ 144 Å.6 and c ϭ 271.0 Å, with two molecules in the asymmetric unit (V m ϭ 6.39 Å 3