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

Biochemistry. 2012 Jan 10;51(1):466-74. doi: 10.1021/bi201539j. Epub 2011 Dec 22.

Abstract

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. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.

MeSH terms

  • Adenosine / chemistry
  • Adenosine / genetics
  • Adenosine / metabolism
  • Amino Acid Sequence
  • Catalytic Domain / genetics
  • Crystallography, X-Ray
  • DNA Methylation
  • Escherichia coli K12 / enzymology*
  • Escherichia coli K12 / genetics
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Humans
  • Methyltransferases / chemistry*
  • Methyltransferases / deficiency
  • Methyltransferases / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Binding / genetics
  • Ribosome Subunits, Small, Bacterial / enzymology
  • Ribosome Subunits, Small, Bacterial / genetics
  • Substrate Specificity / genetics

Substances

  • Escherichia coli Proteins
  • Methyltransferases
  • 16S rRNA (adenine(1518)-N(6)-adenine(1519)-N(6))-dimethyltransferase
  • Adenosine

Associated data

  • PDB/1AQJ
  • PDB/1QAM
  • PDB/1QOS
  • PDB/1QYR
  • PDB/1YFL
  • PDB/1ZQ9
  • PDB/3GRR
  • PDB/3TPZ