Methyltransferases as bioorthogonal labelling tools for proteins

  • Angelica Jimenez Rosales

Student thesis: Phd


Development of enzymatic labelling methods has been driven by the importance of studying molecular structures and interactions to comprehend cellular processes. Methyltransferases (MTases), which regulate genetic expression by transferring a methyl group from the cofactor S-adenosyl-L-methionine (SAM) to DNA, histones and various proteins, have been shown to accept SAM analogues with an alternative alkyl group on the sulfonium centre. These alkyl groups can be transferred to the substrate, and with a further reaction can be selectively functionalized. Thus, MTases together with SAM analogues have emerged as novel labelling tools.The project aims to use MTases to obtain an orthogonal system that can selectively use a SAM cofactor analogue to transfer functional chains to proteins with a specific motif. To achieve selectivity of the system, the SAM analogue cofactor was modified on the ribose ring; to obtain a new transferase activity of the system, the transferable methyl on the sulfonium centre was changed to a different substituent. SAM analogues were produced enzymatically with hMAT2A by using 3'-deoxy-ATP and methionine or ethionine. Mutants of SET8 and novel substrates were designed to have modifications at residues in the active site, within the vicinity of the ribose ring of SAM, and were assessed for selective activity with the new analogue cofactor. The results showed that the new cofactor 3'-deoxy-S-adenosyl-L-methionine (3'dSAM) was efficient in the mono-methylation of the substrate peptide RFRKVL, and that the mutant SET8 C270V exhibited over 13 fold MTase activity in presence of 3'dSAM and the RFRKVL substrate, in comparison with the activity with the WT sequence RHRKVL and the SAM cofactor.In addition, glutathione S-transferase (GST) was used as a model protein to express the motif RFRKVL, to transform it into a potential substrate for SET8. Assessment of the MTase activity of SET8, 3'dSAM and the novel GST substrate indicated mono-methylation of the substrate. Moreover, the motif showed no interference with GST native activity.Based on the observations, a new enzymatic system shows higher selectivity with a new analogue cofactor over SAM to effectively methylate proteins expressing the consensus RFRKVL. Work on substrates, enzymes and cofactors should continue to obtain a functional-chain transferase activity of the enzymatic system.
Date of Award1 Aug 2016
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJason Micklefield (Supervisor)


  • protein labelling
  • SAM analogues
  • SET8
  • methyltransferases
  • analogue cofactor

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