Protein arginine methyltransferases (PRMTs) aid in the regulation of many biological processes. Accurate control of PRMT activity includes recognition of specific arginyl groups within targeted proteins, and also the generation of the correct level of methylation, none of which are fully understood. The predominant PRMT in vivo, PRMT1, has wide substrate specificity and is capable of both mono- and dimethylation, which can induce distinct biological outputs. What regulates the specific methylation pattern of PRMT1 in vivo is unclear. We report that PRMT1 methylates a multisite peptide substrate in a non-stochastic manner, with less C-terminal preference, consistent with the methylation patterns observed in vivo. With a single targeted arginine, PRMT1 catalyzed the dimethylation in a semi-processive manner. The degree of processivity is regulated by substrate sequences. Our results identify a novel substrate-induced mechanism for modulating PRMT1 product specificity. Considering the numerous physiological PRMT1 substrates, as well as the distinct biological outputs of mono- and dimethylation products, such fine-tuned regulation would significantly contribute to the accurate product specificity of PRMT1 in vivo and the proper transmission of biochemical information.
"In summary, we provide a detailed hypothesis of arginine asymmetric dimethylation catalyzed by PRMT1 and discuss the charge distribution and proton transfer process in detail. However, the catalytic mechanism of PRMTs requires further exploration to answer certain questions, such as those on product specificity . Further understanding the PRMT1 catalytic mechanism will be beneficial for the rational design of inhibitors with both efficiency and specificity. "
[Show abstract][Hide abstract] ABSTRACT: Protein arginine methyltransferase 1 (PRMT1), the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet) as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical SN2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.
PLoS ONE 08/2013; 8(8):e72424. DOI:10.1371/journal.pone.0072424 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein arginine methyltransferases (PRMTs) have emerged as attractive therapeutic targets for heart disease and cancers. PRMT5 is a particularly interesting target because it is overexpressed in blood, breast, colon and stomach cancers, and promotes cell survival in the face of DNA damaging agents. As the only known member of the PRMT enzyme family to catalyze the formation of mono and symmetrically dimethylated arginine residues, PRMT5 is also mechanistically unique. As a part of a program to characterize the mechanisms and regulation of the PRMTs, and develop chemical probes targeting these enzymes, we characterized the substrate specificity, processivity, and kinetic mechanism of this enzyme. In this report, we demonstrate that distal positively charged residues contribute to substrate binding in a synergistic fashion. Additionally, we show that PRMT5 catalyzes symmetric dimethylation in a distributive fashion. Finally, the results of initial velocity, product and dead-end inhibition studies indicate that PRMT5 uses a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes. In total, these studies will guide PRMT5 inhibitor development and lay the foundation for studying how the activity of this medically relevant enzyme is regulated.
[Show abstract][Hide abstract] ABSTRACT: Recently peptide-based inhibitors have been used to selectively inhibit a family of epigenetic enzymes called protein arginine N-methyltransferases (PRMTs), which has been implicated in different physiological processes and human diseases, such as heart disease and cancer. The diverse efforts to tease out subtle structural differences among PRMT enzymes in order to generate selective inhibitors as well as existing challenges in the field will be examined. The acquisition of PRMT substrate sequence preferences and structural information obtained from small-molecule inhibitors have helped in developing different peptide-based inhibitors that show great promise not only as inhibitors, but also as molecular probes to characterize PRMTs.
Lauren Sartor, Charmaine Ibarra, Ahmad Al-Mestarihi, Brian O. Bachmann, Jessica L. Vey
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