Characterisation and engineering of the cobalamin-dependent Radical SAM methyltransferase Orf29 for the synthesis of novel SAM derivatives

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The unusual, cyclopropane-containing amino acid (1S,2S)-2-methyl-1-aminocyclopropane-1-carboxylic acid ((1S,2S)-MeACC) is biosynthesised in a two-step enzymatic process that utilises S-adenosyl-L-methionine (SAM) as initial substrate. In the first step, a cobalamin-dependent Radical SAM methyltransferase (B12-RSMT), termed Orf29, catalyses the methylation of the methionine moiety of SAM yielding (4’’R)-4’’-methyl-SAM. In the second step, the MeACC synthase Orf30 is responsible for the cyclisation reaction leading to formation of (1S,2S)-MeACC. In this project, the B12-RSMT Orf29 will be characterized in order to elucidate the structure-function relationship of this intriguing enzyme, which utilizes SAM in three distinct roles: as cofactor for initiation of radical catalysis, as methyl group donor and as substrate. In order to understand the catalytic mechanism of Orf29, we aim to elucidate the sequence of individual reaction steps and define the binding sites for SAM in the active site, which allow the bound SAM to fulfil its distinct roles. Besides the functional and structural characterization of Orf29, we aim to use this enzyme as a platform to synthesize diverse SAM derivatives. For this purpose, biochemical, structural and bioinformatic approaches will be used. Finally, Orf29 variants may be used in the future for the bioorthogonal synthesis of SAM derivatives for incorporation into novel, modified peptide antibiotics.