Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

Abstract

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

Insights into Folate/FAD-dependent tRNA Methyltransferase Mechanism

“…However, these two domains are oriented differentially in the two enzymes. Moreover, several mobile segments form part of the FAD binding site in GidA, and several residues important for tRNA binding are located in these loops (54). Altogether, this suggests that large domain motion may occur during catalysis both in GidA and TrmFO.…”

“…Notably, tRNA and/or MnmE binding coupled to GTP hydrolysis could trigger this conformational rearrangement (51). Interestingly, limited trypsinolysis of GidA suggested that significant conformational changes also occur upon FAD binding (54). The thiol group from one of these catalytically important cysteines was proposed to attack the C6 atom of the target uridine to form a covalent intermediate and subsequently activate the C5 position (51).…”

Stabilization of G Domain Conformations in the tRNA-modifying MnmE-GidA Complex Observed with Double Electron Electron Resonance Spectroscopy

“…In the absence of this modification, which can be due to mutation of one of the two enzymes, neurological disorders such as myoclenic epilepsy ragged red fibers, mitochondrial encephalomyopathy lactic acidosis stroke, and nonsyndromic deafness occur (14 -18). The communication between MnmE and GidA is mandatory for both in vitro and in vivo activity of the complex (4,28,33,34), and although structural information is available for MnmE from different bacterial organisms and EcGidA, the exact role of the two proteins and specifically their interplay in the modification reaction remain largely unknown (1,29,33,35).…”

Bacterial tRNA Modification Enzymes: Potential Role in Biology and Virulence

“…Their study also showed identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from gidA and mnmE mutants suggesting GidA and MnmE form a functional complex in which both proteins are interdependent. Further studies done in E. coli have provided additional evidence suggesting the in vitro binding ability of GidA and MnmE and that together these two enzymes are responsible for the proper biosynthesis of mnm 5 s 2 u in bacterial tRNA [11][12][13]. Additionally, Shippy et al [14] showed that GidA and MnmE bind together to modify Salmonella tRNA.…”