1. Thesis abstract
The University of Manchester
Angélica Jiménez-Rosales
Degree Doctor of philosophy (PhD) in the Faculty of Engineering and Physical Sciences
Methyltransferases for protein labelling and immobilisation
February 2016
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.