Investigation of PRMT1 residues which modulate activity & control TypeI/TypeII dimethylation

Protein arginine methyltransferases (PRMTs) catalyze methyl group transfer from S‐adenosyl methionine to a guanidino nitrogen of a protein arginine residue creating monomethyl arginine (MMA) and S‐adenosyl homocysteine. TypeI methyltransferases, PRMT1, 2, 3, 4, 6, and 8, dimethylate arginine asymmetrically on the same guanidino nitrogen and form ADMA. TypeII PRMTs (5 and 7), perform symmetric dimethylation (SDA) where both guanidino nitrogens are modified. In some instances, SDA or ADMA are found at the same amino acid position and appear to have opposing biological consequences. Also, proteolysis of modified proteins releases MMA, SDA, and/or ADMA. ADMA, an endogenous inhibitor of nitric oxide synthase, is a proposed cardiovascular and endothelial dysfunction risk factor. Thus, understanding how the PRMTs control Type1 versus TypeII dimethylation is important. Based on the PRMT1 crystal structure and the PRMT5 protein sequence, Met 155 of PRMT1 was hypothesized to affect dimethylation Type by preventing free rotation about the terminal C‐N bond of the substrate arginine in the PRMT1 active site, thus only forming ADMA. In order to test this theory, PRMT1M155A was purified and the activity was assessed using the 19 amino acid peptide R3 derived from the in vivo PRMT1 substrate, fibrillarin. (M155A Vmax= 3 μM/min, WT Vmax= 11 μM/min) Hydrolyzed amino acids from the mutant catalyzed peptide product were analyzed by fluorescent derivatization via RP‐HPLC. Results showed that MMA and ADMA were produced. Thus, removing steric bulk afforded by M155 in the active site of PRMT1 is not by itself sufficient to transform PRMT1 into a TypeII PRMT. Funded by USU New Faculty Funds