Characterization of the Staphylococcus aureus rRNA methyltransferase encoded by orfX, the gene containing the staphylococcal chromosome Cassette mec (SCCmec) insertion site.

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.

Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis.

Control of substrate specificity by a single active site residue of the KsgA methyltransferase.

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine.

Binding of adenosine-based ligands to the MjDim1 rRNA methyltransferase: implications for reaction mechanism and drug design.

The KsgA/Dim1 family of proteins is intimately involved in ribosome biogenesis in all organisms. These enzymes share the common function of dimethylating two adenosine residues near the 3'-OH end of the small subunit rRNA; orthologs in the three kingdoms, along with eukaryotic organelles, have evolved additional functions in rRNA processing, ribosome assembly, and, surprisingly, transcription in mitochondria. The methyltransferase reaction is intriguingly elaborate.

Structural and functional divergence within the Dim1/KsgA family of rRNA methyltransferases.

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported.

Sequence and structural evolution of the KsgA/Dim1 methyltransferase family.

Background: One of the 60 or so genes conserved in all domains of life is the ksgA/dim1 orthologous group. Enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. Despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions.

A conserved rRNA methyltransferase regulates ribosome biogenesis.

In contrast to the diversity of most ribosomal RNA modification patterns and systems, the KsgA methyltransferase family seems to be nearly universally conserved along with the modifications it catalyzes. Our data reveal that KsgA interacts with small ribosomal subunits near functional sites, including Initiation factor 3 and 50S subunit binding sites. These findings suggest a checkpoint role for this modification system and offer a functional rationale for the unprecedented level of conservation.

Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution.

Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps.

Crystal structure of KsgA, a universally conserved rRNA adenine dimethyltransferase in Escherichia coli.

The bacterial enzyme KsgA catalyzes the transfer of a total of four methyl groups from S-adenosyl-l-methionine (S-AdoMet) to two adjacent adenosine bases in 16S rRNA. This enzyme and the resulting modified adenosine bases appear to be conserved in all species of eubacteria, eukaryotes, and archaebacteria, and in eukaryotic organelles. Bacterial resistance to the aminoglycoside antibiotic kasugamycin involves inactivation of KsgA and resulting loss of the dimethylations, with modest consequences to the overall fitness of the organism.

Crystallization and preliminary X-ray diffraction analysis of KsgA, a universally conserved RNA adenine dimethyltransferase in Escherichia coli.

The bacterial enzyme KsgA catalyzes the transfer of a total of four methyl groups from S-adenosylmethionine (SAM) to two adjacent adenosines in 16S rRNA. These modified adenosines are universally conserved in all species of eubacteria, eukaryotes and archaebacteria studied. Recombinant KsgA from Escherichia coli was overexpressed as a His-tagged fusion protein and purified.