Project Details
Projekt
Enzymatic generation of double-modified SAM analogues
Applicant
Professorin Dr. Andrea Rentmeister
Subject Area
Biological and Biomimetic Chemistry
Term
since 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 510974120
Methionine adenosyltransferases (MATs) produce S-adenosyl-L-methionine (SAM) from methionine and ATP. SAM serves as a cosubstrate for most methyltransferases (MTs), however many of them are promiscuous with regard to the methyl (or alkyl or benzyl) group at the sulfonium centre. The widespread occurrence of MTs prevents the use of SAM analogues with extended moieties at the sulfonium centre for studying individual MTs or labelling biomolecules selectively in cellular systems. We propose to generate double-modified SAM analogues with modifications at the sulfonium centre and the adenosine part of SAM. The combined modifications shall prevent the conversion by wildtype MTs. We aim to generate these double-modified SAM analogues enzymatically by engineering PC-MjMAT, a MAT variant from Methanocaldococcus jannaschii with high activity on benzylic methionine analogues and a number of ATP analogues. Specifically, we plan to engineer PC-MjMAT variants to use ortho-nitrobenzyl-homocysteine with GTP or 2 phenylethynyl-ATP by first applying site-directed mutagenesis and then directed evolution. The new MAT variants will subsequently be tested in cascade reactions with MTs from our group and other FOR 5596 partners. We will begin with site-directed mutations to enhance two MTs (NovO and MTaqI) toward the double-modified SAM analogues in the final stage of this grant period. Ultimately, we aim to create bioorthogonal SAM analogues to achieve conversion by a specific MT variant and thus selective modifications in cellular systems. The ability to selectively modify a target molecule in cells would open a new way for biomolecular labelling and - in combination with the photo-cleavable groups, such as the ortho-nitrobenzyl moiety - even for controlling biomolecular functions (e.g. in epigenetics) in the complex cellular environment.
DFG Programme
Research Units
