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 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 nonstochastic 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 semiprocessive 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.

Protein-arginine methyltransferases aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is due in part to the diverse product specificity displayed by the protein-arginine methyltransferases. In addition to site location within a protein, biological response is also determined by the degree (mono-/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here, we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity but also control substrate specificity. Mutation of Met-155 or Met-48 results in a loss in activity and a change in distribution of mono-and dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is much slower than transfer of the first methyl group in M48L, especially for arginine residues located in the center of the peptide substrate where turnover of the monomethylated species is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.Protein methylation is a significant post-translational modification in eukaryotic organisms. Protein arginine residues can be methylated on the guanidino nitrogens by protein-arginine methyltransferases (PRMTs), 2 which use S-adenosyl-L-methionine (AdoMet) as a methyl group donor. This post-translational modification is important in a wide variety of fundamental biological processes, including transcription, RNA splicing, signal transduction, DNA repair, viral replication (reviewed in Ref. 1), and chromatin remodeling (2). In recent years, the significance of PRMTs in human diseases has been increasingly studied, especially in cardiovascular disease (3) and cancer (4). In all, PRMTs play a crucial role in many biological processes.Although the biological importance of PRMTs has become well accepted, the current knowledge of the fundamental biochemistry of these enzymes is limited, due in part to the complexity of the system. So far, 11 PRMT isoforms have been identified. In mammalian cells, nine PRMTs catalyze monomethyl arginine (MMA) formation, and they can be categorized into two major types as follows: PRMT1, -2-4, -6, and -8 additionally catalyze asymmetric dimethyl arginine (ADMA) formation, demonstrating type I activity; and PRMT5, -7, and -9 catalyze symmetric dimethyl arginine (SDMA) formation, demonstrating type II activity (Fig. 1A). PRMT10 and -11 were identified as putative PRMT genes with no methylation activity shown as yet (5). As with other enzyme f...

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Background:Oxidative stress leads to increased PRMT1 expression and ADMA accumulation. Results: PRMT1 activity is increased by reductants and decreased by oxidants. Conclusion: PRMT1 activity is regulated in a redox-sensitive manner. Significance: The role of PRMT1 activity in the oxidative stress response may be more complex than previously thought.

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs), and is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Reports have shown that DDAH activity is down regulated and PRMT1 protein expression is upregulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. Mutagenesis studies demonstrate that the oxidation event involves two cysteine residues on PRMT1. We demonstrate a hydrogen peroxide concentration‐dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. We additionally show that PRMT1 purified from oxidatively challenged human cells displays impaired activity compared to PRMT1 purified from healthy human cells. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis, but rather demonstrate that enzymatic activity can be regulated in a redox‐sensitive manner. Our findings suggest that modulation of protein arginine methylation could play a large role in the oxidative stress response.