Mechanism of Multiple Lysine Methylation by SET Domain Enzyme Rubisco LS

In 2000, the SET domain was identified as a ~110 amino acid motif shared by chromatin structure and gene silencing-related proteins histone H3 ε N-methyltransferases (HMTs) and Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) Large Subunit ε Nmethyltransferases (LSMT). SET domain protein methyltransferases catalyze the transfer of methyl groups from the cofactor S-adenosylmethionine (AdoMet) to specific lysine residues of protein substrates, such as the N-terminal tails of histones H3 and H4 and the LS of the Rubisco holoenzyme complex. In 2003, a crystal structure of Rubisco LSMT with free Lysine bound to the active site was obtained. In order to determine function, I investigated the possibility of Lysine as an alternative substrate and competitive inhibitor with respect to Rubisco. Through kinetic analysis, I found that free lysine and monomethyllysine does act as a competitive inhibitor, and their Kis are 36.6 mM and 100.8 mM. Furthermore, the products of a radiometric assay with lysine, monomethyllysine, dimethyllysine as a substrate were analyzed using TLC and lysine, monomethyllysine and dimethylysine were used as substrates. The structural data concluded that the methyl group of MeLys is bound to the enzyme via carbon-oxygen hydrogen bonds that play a key role in catalysis. The methyl donor and the acceptor are aligned in a linear geometry for SN2 nucleophilic transfer of methyl group during catalysis. The functional data and structural data gave insight into catalytic mechanism of multiple lysine methylation and are included in a July 2003 Nature Structural Biology paper