4FAK

Crystal Structure of OrfX in Complex with S-Adenosylmethionine

Summary for 4FAK

• Entry DOI: 10.2210/pdb4fak/pdb
• Descriptor: Ribosomal RNA large subunit methyltransferase H, S-ADENOSYLMETHIONINE, TETRAETHYLENE GLYCOL, ... (5 entities in total)
• Functional Keywords: alpha/beta methyltransferase rossmann fold, rrna methylation, adomet, rrna, transferase, ribosomal protein
• Biological source: Staphylococcus aureus
• Cellular location: Cytoplasm (Potential): P0C1V0
• Total number of polymer chains: 1
• Total formula weight: 19440.45

Authors

Safo, M.K.,Musayev, F.N.,Boundy, S.,Archer, G.L.,Rife, J.P.,O'Farrell, H.C. (deposition date: 2012-05-22, release date: 2012-06-06, Last modification date: 2023-09-13)

Primary citation

Boundy, S.,Safo, M.K.,Wang, L.,Musayev, F.N.,O'Farrell, H.C.,Rife, J.P.,Archer, G.L.
Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX, the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site.
J.Biol.Chem., 288:132-140, 2013

PubMed Abstract: The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.

PubMed: 23150671
DOI: 10.1074/jbc.M112.385138

Experimental method

X-RAY DIFFRACTION (1.7 Å)