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Two protein lysine methyltransferases methylate outer membrane protein B from Rickettsia

Amila H Abeykoon1, Chien-Chung Chao, Guanghui Wang

  • 1Department of Chemistry, Georgetown University, Washington, DC, USA.

Journal of Bacteriology
|September 25, 2012
PubMed

Insights

Researchers identified and characterized two novel lysine methyltransferases, rRP789 and rRP027-028, from Rickettsia prowazekii. These enzymes are crucial for methylating outer membrane protein B (OmpB), impacting bacterial virulence and offering new avenues for diagnostics and vaccines.

Area of Science:

  • Microbiology
  • Biochemistry
  • Molecular Biology

Background:

  • Rickettsia prowazekii causes epidemic typhus and is a potential biological threat.
  • Outer membrane protein B (OmpB) is a key antigen involved in rickettsial virulence.
  • Lysine methylation of OmpB correlates with virulence, suggesting enzymatic activity is critical, yet no such enzymes were previously identified.

Purpose of the Study:

  • To identify and biochemically characterize lysine methyltransferases responsible for OmpB methylation in Rickettsia.
  • To investigate the enzymatic activity and specificity of these novel methyltransferases.

Main Methods:

  • Bioinformatic analysis of Rickettsia genomes to identify putative methyltransferases.
  • Gene synthesis, cloning, and expression in E. coli.
  • Protein purification using Ni-NTA affinity chromatography.
  • Enzyme activity assays using radioactively labeled S-adenosylmethionine and recombinant OmpB fragments.
  • Western blot analysis and liquid chromatography-tandem mass spectrometry (LC/MS-MS) for methylation analysis.

Main Results:

  • Two recombinant methyltransferases, rRP789 and rRP027-028, were successfully purified and demonstrated methyltransferase activity on OmpB fragments.
  • rRP789 showed 10-30 fold higher specific activity than rRP027-028.
  • Both enzymes catalyzed trimethylation, with rRP789 also capable of mono- and dimethylation, while rRP027-028 exclusively performed trimethylation.
  • These are the first characterized lysine methyltransferases for outer membrane proteins in Gram-negative bacteria.

Conclusions:

  • rRP789 and rRP027-028 are the first identified and characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria.
  • These findings provide essential tools for studying OmpB methylation mechanisms, structure-function relationships, and for developing novel diagnostic assays and vaccine candidates against Rickettsia prowazekii.

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