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.

Les protéines arginine méthyl transférases ("PRMTs") sont impliquées dans de nombreux processus cellulaires essentiels. La protéine CARMI ("Coactivator-associated arginine methyltransferase 1", appelée aussi "PRMT4") a été initialement identifiée par sa fonction co-activatrice de la transcription impliquantplusieurs récepteurs nucléaires des hormones. CARMI est une enzyme qui catalyse la réaction de méthylation sur les histones via un donneur de méthyl naturel, la S-adénosY-L -méthionine (SAM). De nombreux travaux ont montré que CARMI est surexprimée dans les cancers du sein et de la prostate. L' objectif de ce travail est la compréhension à l'échelle moléculaire du mode d'action de CARMI et l'étude du mécanisme de reconnaissances moléculaires et de transferts d' informations gouvernés par la protéine CARMI. La structure cristallographique obtenue de cette enzyme en présence de cofacteur, la S-AdénosyhHomocystéine ou la Sinefungine a eu un effet stabilisant. Ainsi, notre stratégie a été de créer des molécules hameçons basées sur le motif de la SAM capables d' ancrer un peptide mimant la séquence de l' histone H3, pour ensuite les tester en co-cristallisation avec CARMI. Ainsi, grâce à la diffraction aux rayons X, les interactions mises en jeu dans le complexe CARMlImolécules hameçons/peptide pourront être déterminées. Cette stratégie s'est effectuée en trois étapes : la première étape, décrite dans le chapitre 2, a consisté en la synthèse d'analogues de la SAM obtenus grâce à des modifications réalisées autour de l'atome de soufre. Ces composés nous ont permis d' explorer la « poche du sulfonium ». Puis la seconde étape, décrite dans le chapitre 3, a été la synthèse • d'analogues de bisubstrats nécessaires pour l'exploration de la « poche de l'arginine ». Dans une dernière étape, décrite dans les chapitres 4 et 5, nous avons abordé la synthèse d'adduits SAM-peptide pouf pouvoir étudier le « domaine de fixation du peptide ». Dans le quatrième chapitre, la méthode de choix est la création d'un lien covalent entre une molécule hameçon électrophile etun peptide par chimie de click in-situ : par réaction de cycloaddition de Huisgen; par réaction entre des molécules hameçons électrophiles capables de piéger un peptide cystéine ou un peptide arginine. Ces essais se sont révélés infructueux et une nouvelle stratégie a été employée en utilisant des molécules ancres. Dansle cinquième chapitre des molécules ancres ont donc été préformés pour ensuite être testés en cocristallisation dans CARMl. / Protein aginine methyltransferases (PRMTs) have been implicated in a variety of biological processes. Coactivator-associated arginine methyltransferase 1 (CARM1 , also known as PRMT4) was identified as an enhancer of the transcriptional activation by several nuclear hormone receptors CARM1 is an enzyme which methylates the arginines of histones via a natural methyl donor, the SAdenosyh-Methionine (SAM). Recent studies have shown that CARM-1 is over-expressed in breastumors and in hormone dependent prostate tumors. The goal of this work is to understand at the molecular level the mode of binding of substrate/product arginine-containing peptides, reflectingstates prior and subsequent to methylation and the detailed mechanism of action of this protein. Several crystal structures of the catalytic domain of CARM1 have shown that cofactor-binding, such as S-Adenosyl-L -Homocysteine or sinefungin, produces large conformational changes in the catalytic domain. These crystal structures clearly illustrate that SAM binding is a prerequisite for peptide binding and build up the productive peptide binding site. Our strategy was to design fishhook molecules derivatives of the SAM capable of anchoring a mimic peptide of the histone H3 in order to test the co-crystallization in CARM1. Consequently, thanks to X-Ray structure, interactions involvement in the complexe CARM1/fishhook molecule/peptide could be determined. This strategy was do ne in three steps: the first one, described in the chapter 2, consisted in synthesizing SAManalogues with several modifications around sulfur atom. These compounds permitted to explore the "sulfonium pocket". The second step, described in chapter 3, consisted in synthesizing analogues of bisubstrats to explore "arginine binding pocket". Finally, the last step, described in the chapter 4 and 5, consisted in synthesizing SAM-peptide adducts to study "peptide binding domain". ln the chapter 4, the chosen method is the creation of covalent link between this molecule and a peptide by click chemistry in-situ: by reaction of Huisgen's cycloaddition; by reaction between electrophilic fishhook molecules capable of capturing with a cystein or arginine peptides. Unfortunately, ail of these trials have been unsuccessful. Consequently SAM-peptide adducts were performed to be co-crystallized in CARM1. This part was described in the last chapter.