ELUCIDATING PRODUCT SPECIFICITY AND DETERMINING THE ACTIVE SITE BASE IN PROTEIN ARGININE METHYLTRANSFERASE 7 (PRMT7) USING QM/MM/MD AND EXPERIMENT

Abhishek Thakur,¹ Betsy Caceres,² Joan M. Hevel,² and Orlando Acevedo¹

1Department of Chemistry, University of Miami, Coral Gables, FL 33146.
2Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322.

Protein arginine methylation is an important post-translational modification involved in cellular processes, RNA processing, and gene transcription. The arginine methylation enzyme of interest, protein arginine methyltransferase 7 (PRMT7), exclusively generates the first turnover product, monomethylated arginine (MMA), but neither of the second turnover products, asymmetric dimethylated arginine (ADMA) and symmetric dimethylated arginine (SDMA). However, it remains unclear how PRMT7 product specificity is regulated and which active site base governs the proton abstraction in the S_N2 mechanism. Experimental mutagenesis work has been performed and it was determined that three different single amino acid mutations, i.e., Phe71Ile, Met75Leu, and Glu181Asp, in the PRMT7 active site can enable the enzyme to perform the second turnover and that Glu172 might be acting as the base. Combined quantum mechanical and molecular mechanical calculations and molecular dynamics simulations (QM/MM/MD) that employed free energy perturbation methods have been used to help elucidate the origin of the product specificity in both WT and mutant PRMT7 enzymes. The present simulations predicted a lower ΔG^‡ barrier for MMA over ADMA and SDMA and a preference for Glu172 over Glu181 in the proton abstraction step. Additional single and double mutations, e.g., Met75Phe, Glu181Asp-Gln329Ser, and Met75Leu-Gln329Ser, for PRMT7 were investigated and discussed.