Mutational Analysis Defines the Roles of Conserved Amino Acid Residues in the Predicted Catalytic Pocket of the rRNA:m6A Methyltransferase ErmC′

Maravić, Gordana; Feder, Marcin; Pongor, Sándor; Flögel, Mirna; Bujnicki, Janusz M.

doi:10.1016/s0022-2836(03)00863-5

Cited by 36 publications

(46 citation statements)

References 41 publications

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“…After donating the methyl group, AdoMet is converted into AdoHcy (12). Sequence alignment and structure superimposition of fibrillarin with other 2Ј-O-methylation MTases revealed a conserved KDK triad at the site of methyl transfer (28). Substitution of these three residues in the bacterial MTase RrmJ with alanine had deleterious effects on its methylation activity toward the 23 S rRNA substrate (23).…”

Section: Discussionmentioning

confidence: 99%

Structural and Thermodynamic Evidence for a Stabilizing Role of Nop5p in S-Adenosyl-L-methionine Binding to Fibrillarin

Aittaleb¹,

Visone²,

Fenley

2004

Journal of Biological Chemistry

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In Archaea, fibrillarin and Nop5p form the core complex of box C/D small ribonucleoprotein particles, which are responsible for site-specific 2 -hydroxyl methylation of ribosomal and transfer RNAs. Fibrillarin has a conserved methyltransferase fold and employs S-adenosyl-L-methionine (AdoMet) as the cofactor in methyl transfer reactions. Comparison between recently determined crystal structures of free fibrillarin and fibrillarinNop5p-AdoMet tertiary complex revealed large conformational differences at the cofactor-binding site in fibrillarin. To identify the structural elements responsible for these large conformational differences, we refined a crystal structure of Archaeoglobus fulgidus fibrillarin-Nop5p binary complex at 3.5 Å. This structure exhibited a pre-formed backbone geometry at the cofactor binding site similar to that when the cofactor is bound, suggesting that binding of Nop5p alone to fibrillarin is sufficient to stabilize the AdoMet-binding pocket. Calorimetry studies of cofactor binding to fibrillarin alone and to fibrillarin-Nop5p binary complex provided further support for this role of Nop5p. Mutagenesis and thermodynamic data showed that a cationbridge formed between Tyr-89 of fibrillarin and Arg-169 of Nop5p, although dispensable for in vitro methylation activity, could partially account for the enhanced binding of cofactor to fibrillarin by Nop5p. Finally, assessment of cofactor-binding thermodynamics and catalytic activities of enzyme mutants identified three additional fibrillarin residues (Thr-70, Glu-88, and Asp-133) to be important for cofactor binding and for catalysis.

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Section: Discussionmentioning

confidence: 99%

Structural and Thermodynamic Evidence for a Stabilizing Role of Nop5p in S-Adenosyl-L-methionine Binding to Fibrillarin

Aittaleb¹,

Visone²,

Fenley

2004

Journal of Biological Chemistry

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“…The methylation of the N 6 position of adenine has been investigated in DNA (for structural studies see [42][43][44] ) and more recently in rRNA. 45,46 Interestingly, the main catalytic residue in m 6 which is proposed to stabilize the cationic transition state by cation-π interaction. 49,50 And the second one is the aromatic residue Y/F/W of motif VIII, which stabilizes the flipped base outside the DNA helix.…”

Section: Superimposition Of T Thermophilus and M Tuberculosismentioning

confidence: 99%

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

Barraud

Golinelli‐Pimpaneau

Atmanène

et al. 2008

Journal of Molecular Biology

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“…Dissociation constants for RsmD complexes with unmethylated 30S subunits were found to be in the 30-40 nM range. This showed that binding is approximately two orders of magnitude better than those demonstrated for comparable rRNA methyltransferases RsmC (Sunita et al 2007), Sgm (Husain et al 2010), RlmI (Sunita et al 2008), and ErmC (Maravic et al 2003). The Michaelis constant of RsmD toward the 30S subunit is much better than that of comparable rRNA methyltransferases, while kcat is comparable to other enzymes of this type.…”

Section: Discussionmentioning

confidence: 87%

“…RrmJ, which is responsible for methylation of the 29-OH of nucleotide U2552 of the 23S rRNA, displays a K m of 0.7 mM relative to the 50S subunits and kcat of 0.064 min À1 (Hager et al 2002). The methyltransferase ErmC, which has a kcat of 0.066 sec À1 and K m of 2.8 mM, provides erythromycin resistance (Maravic et al 2003). RsmD methyltransferase has a kcat of 0.028 6 0.004 min À1 and K m of 3.3 6 0.6 nM.…”

Section: Discussionmentioning

confidence: 99%

“…Peptide motifs (D/E)x(G/F)xGxG and (N/D/S)PP(Y/F/W/ H), characteristic for all methyltransferases of this class, could be found in the RsmD primary structure ( 58 DxFxGxG and 127 DPPF). In a substantial number of examples described in the literature, even single amino acid substitutions of the conserved active site residues resulted in the complete loss of enzymatic activity (Hager et al 2002;Maravic et al 2003). However, conserved proline residues PP128-129 of RsmD could be substituted for alanines without catalytic activity loss.…”

Section: Discussionmentioning

confidence: 99%

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Properties of small rRNA methyltransferase RsmD: Mutational and kinetic study

Sergeeva

Prokhorova²,

Ordabaev³

et al. 2012

RNA

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Ribosomal RNA modification is accomplished by a variety of enzymes acting on all stages of ribosome assembly. Among rRNA methyltransferases of Escherichia coli, RsmD deserves special attention. Despite its minimalistic domain architecture, it is able to recognize a single target nucleotide G966 of the 16S rRNA. RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit. Here, we show that it possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit modified at G966. RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunit's involvement in translation.

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Mutational Analysis Defines the Roles of Conserved Amino Acid Residues in the Predicted Catalytic Pocket of the rRNA:m6A Methyltransferase ErmC′

Cited by 36 publications

References 41 publications

Structural and Thermodynamic Evidence for a Stabilizing Role of Nop5p in S-Adenosyl-L-methionine Binding to Fibrillarin

Structural and Thermodynamic Evidence for a Stabilizing Role of Nop5p in S-Adenosyl-L-methionine Binding to Fibrillarin

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

Properties of small rRNA methyltransferase RsmD: Mutational and kinetic study

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