Functional-Structural Analysis of Threonine 25, a Residue Coordinating the Nucleotide-bound Magnesium in Elongation Factor Tu

Krab, Ivo M.; Parmeggiani, Andrea

doi:10.1074/jbc.274.16.11132

Cited by 7 publications

(8 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The catalysis of both mutants is comparably enhanced by kirromycin, but ribosomes stimulate EF-Tu[T61A] much less than EF-Tu[T61N], which is activated to an extent approaching that of wt. This provides more evidence that GTPase activation by kirromycin and ribosomes follows different mechanisms (22). The low ribosome stimulation of the EF-Tu[T61A] GTPase we find here was not observed for T. thermophilus EF-Tu Tt [T62A] (20) at an assay temperature of 37°C that is suboptimal for the thermophilic enzyme.…”

Section: Discussionmentioning

confidence: 65%

“…Since EF-Tu[I60A] has no direct contacts with either nucleotide or magnesium, a local conformation disturbance seems the most likely explanation for its higher GTP dissociation rate. The similar and remarkably modest effect of the Thr-61fAla mutation might seem to be in contrast with the prominence of the Thr-61-Mg 2+ ion link in the crystal structures, also when compared to the drastic change caused by mutation of the opposite Thr-25 to alanine (22). However, this finding agrees with experimental evidence that in H-ras p21 the coordination of the homologous residue Thr-35 to magnesium is rather weak (34,35), despite the presence of a similarly prominent interaction in the crystal.…”

Section: Discussionmentioning

confidence: 90%

“…Construction, OVerproduction, and Purification of EF-Tu Mutants. For rapid purification purposes wt EF-Tu and mutants were overproduced as fusion with glutathione S-transferase and purified as described before (22). Mutations were introduced by the "Unique Site Elimination" method (23), using a kit from Pharmacia.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Mutagenesis of Three Residues, Isoleucine-60, Threonine-61, and Aspartic Acid-80, Implicated in the GTPase Activity of Escherichia coli Elongation Factor Tu

Krab

Parmeggiani

1999

Biochemistry

Self Cite

View full text Add to dashboard Cite

The properties of variants of elongation factor (EF) Tu mutated at three positions implicated in its GTPase activity are presented. Mutation I60A, which reduces one wing of a "hydrophobic barrier" screening off the nucleophilic water molecule found at the GTP gamma-phosphate, causes a reduction of the intrinsic GTPase activity contrary to prediction and has practically no influence on other properties. Mutation D80N, which in the isolated G-domain of EF-Tu caused a strong stimulation of the intrinsic GTPase, reduces this activity in the intact molecule. However, whereas for wild-type EF-Tu complex formation with aa-tRNA reduces the GTPase, EF-Tu[D80N] shows a strongly increased activity when bound to Phe-tRNA. Moreover, ribosomes or kirromycin can stimulate its GTPase up to the same level as for wild-type. This indicates that a local destabilization of the magnesium binding network does not per se cause an increased GTPase but does affect its tight regulation. Interestingly, mutant D80N sequestrates EF-Ts by formation of a more stable complex. Substitutions T61A and T61N induce low intrinsic GTPase, and the stimulation by ribosome is less for T61A than for T61N but still detectable, while kirromycin stimulates the GTPase of both mutants equally. This provides more evidence that stimulation by kirromycin and ribosomes follows a different mechanism. The functional implications of these mutations are discussed in the context of a transition state mechanism for catalysis. An alternative structural explanation for the strong conservation of Ile-60 is proposed.

show abstract

Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 90%

See 1 more Smart Citation

Mutagenesis of Three Residues, Isoleucine-60, Threonine-61, and Aspartic Acid-80, Implicated in the GTPase Activity of Escherichia coli Elongation Factor Tu

Krab

Parmeggiani

1999

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…In this respect, it is worthy to point out that the similar D57A mutation of p21 ras was reported to increase both the dissociation and association rate constants of the protein with GTP, although the effect was greater on the first one (31). An increase in the GTP dissociation rate constant of the MnmE mutant D270A may cause a technical problem concerning the evaluation of its GTP binding capacity by means of the filter binding assay (32). Such an increase could favor partial loss of the nucleotide during filter washing and produce lower B max values.…”

Section: Biochemicalmentioning

confidence: 99%

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Yim¹,

Martínez-Vicente²,

Villarroya³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.The evolutionarily conserved MnmE (TrmE) protein of Escherichia coli is a GTPase that differs extensively from regulatory GTPases such as p21 (1). Thus, MnmE exhibits a very high intrinsic GTPase hydrolysis rate and low affinity for GTP and GDP, and it can form self-assemblies. MnmE has a molecular mass of 50 kDa and is organized as a multidomain protein (see Fig. 1) consisting of an ϳ220-amino acid N-terminal domain, probably required for self-assembly, a middle GTPase domain, of about 160 residues, and an ϳ75-amino acid C-terminal domain, which contains the only Cys residue present in the protein. Strikingly, the isolated GTPase domain roughly conserves the guanine nucleotide binding and GTPase activities of the intact MnmE molecule (1).Null mnmE mutants are defective in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U 34 ) 1 (2), which is found in the wobble position (position 34) of tRNAs that read codons ending with A or G, in the mixed codon family boxes, specifically tRNAs for lysine and glutamic acid (3). The mnmA gene product (6) carries out thiolation in the 2-position of the wobble uridine (U 34 ), whereas mnmE controls the first step of the modification in the 5Ј-position, but it is unclear how many steps precede the formation of cmnm 5 s 2 U 34 (2, 4). Several data support that a second gene, named gidA, trmF, or mnmG, is also involved in the cmnm 5 group addition and that the MnmE activity precedes the activity of GidA (2, 5). The mnmC gene product has two enzymatic activities that transform the cm...

show abstract

“…The different species of EF-Tu were overproduced in E. coli strain DH5a as fusion with glutathione S-transferase [18]. The transformed E. coli strains were grown at 37 C in 2 L of LB rich medium containing 50 mg ml À1 ampicillin to 0.5 A 600 , after which induction with 0.1 mM isopropyl-ß-D-thiogalacto-pyranoside took place at 23 C with incubation up to 17 h. Under these conditions, high level of soluble EF-Tu was obtained.…”

Section: Purification Of Recombinant Frateuria W-315 Ef-tu and Ecolimentioning

confidence: 99%

EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance

2016

View full text Add to dashboard Cite

In this report, we have demonstrated that the poly(U)-dependent poly(Phe) synthesis activity of elongator factor Tu (EF-Tu) from the enacyloxin producing strain Frateuria sp. W-315 is inhibited by the antibiotic similarly to that of Escherichia coli EF-Tu. The inhibitory effect of enacyloxin observed in a purified system was the same as that obtained with an S30 extract from E. coli or Frateuria sp. W-315, respectively, suggesting that antibiotic resistance of enacyloxin producing Frateuria sp. W-315 is not due neither to EF-Tu nor to other components of the translation machinery but to a still unknown mechanism. The EF-Tu gene, as PCR amplified from Frateuria W-315 genomic DNA and sequenced represented an ORF of 1191 nucleotides corresponding to 396 amino acids. This protein is larger than the product of tufA from E. coli by only two amino acid residues. Alignment of the amino acid sequence of EF-Tu from E. coli with those of Frateuria and Ralstonia solanacearum indicates on average 80% identical amino acid residues and 9.7% conservative replacements between EF-Tu Frateuria and EF-Tu E. coli, on one hand, and 97% identity and 1.7% conservative replacement between EF-Tu Frateuria and EF-Tu Ralstonia solanacearum, on the other hand. These strong primary structure similarities between EF-Tu from different origins are consistent with the fact that this factor is essential for the translation process in all kingdoms of life. Comparison of the effects of antibiotics on EF-Tu Frateuria and EF-Tu E. coli revealed that enacyloxin, kirromycin and pulvomycin exert a stronger stimulation of the GDP dissociation rate on EF-Tu Frateuria, while the effects of the antibiotics on the GDP association rate were comparable for the two EF-Tu species. Different mutants of EF-Tu E. coli were constructed with the help of site directed mutagenesis by changing one or several residues of EF-Tu E. coli by the corresponding residues of EF-Tu Frateuria. The single A45K substitution did not modify the intrinsic GTPase activity of EF-Tu E. coli. In contrast, a 2-3 fold stimulation of the intrinsic GTPase activity was observed with the single A42E, F46Y, Q48E and the double F46Y/Q48E substitution. Finally, up to a 7 fold stimulation was observed with the quadruple substitution (mutant A42E/A45K/F46Y/Q48E.

show abstract

Functional-Structural Analysis of Threonine 25, a Residue Coordinating the Nucleotide-bound Magnesium in Elongation Factor Tu

Cited by 7 publications

References 43 publications

Mutagenesis of Three Residues, Isoleucine-60, Threonine-61, and Aspartic Acid-80, Implicated in the GTPase Activity of Escherichia coli Elongation Factor Tu

Mutagenesis of Three Residues, Isoleucine-60, Threonine-61, and Aspartic Acid-80, Implicated in the GTPase Activity of Escherichia coli Elongation Factor Tu

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance

Contact Info

Product

Resources

About