The conserved aspartate in motif III of b family AdoMet-dependent DNA methyltransferase is important for methylation.

S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) are involved in diverse cellular functions. These enzymes show little sequence conservation but have a conserved structural fold. The DNA MTases have characteristic motifs that are involved in AdoMet binding, DNA target recognition and catalysis. Motif III of these MTases have a highly conserved acidic residue, often an aspartate, whose functional significance is not clear. Here, we report a mutational study of the residue in the ß family MTase of the Type III restriction-modification enzyme EcoP15I. Replacement of this residue by alanine affects its methylation activity. We propose that this residue contributes to the affinity of the enzyme for AdoMet. Analysis of the structures of DNA, RNA and protein MTases reveal that the acidic residue is conserved in all of them, and interacts with N6 of the adenine moiety of AdoMet. Interestingly, in the SET-domain protein lysine MTases, which have a fold different from other AdoMet-dependent MTases, N6 of the adenine moiety is hydrogen bonded to the main chain carbonyl group of the histidine residue of the highly conserved motif III. Our study reveals the evolutionary conservation of a carbonyl group in DNA, RNA and protein AdoMet-dependent MTases for specific interaction by hydrogen bond with AdoMet, despite the lack of overall sequence conservation.

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme).

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here, we present the X-ray crystal structure of the TbPRMT1 ENZ-Δ52PRO tetrameric complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.4 Å resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of an ENZ-specific tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRMTs in that it lacks the conserved η2 310-helix within the Rossmann fold, abolishing cofactor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, as heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity toward the substrate protein TbRGG1. Together, we provide a structural basis for TbPRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the TbPRMT1 prozyme, which represents a novel mode of PRMT regulation.

Rational Design, synthesis and biological evaluation of novel triazole derivatives as potent and selective PRMT5 inhibitors with antitumor activity.

Protein arginine methyltransferase 5 (PRMT5) is responsible for the mono-methylation and symmetric dimethylation of arginine, and its expression level and methyl transferring activity have been demonstrated to have a close relationship with tumorigenesis, development and poor clinical outcomes of human cancers. Two PRMT5 small molecule inhibitors (GSK3326595 and JNJ-64619178) have been put forward into clinical trials. Here, we describe the design, synthesis and biological evaluation of a series of novel, potent and selective PRMT5 inhibitors with antiproliferative activity against Z-138 mantle cell lymphoma cell line. Among them, compound C_4 exhibited the highest potency with enzymatic and cellular level IC50 values of 0.72 and 2.6 µM, respectively, and displayed more than 270-fold selectivity toward PRMT5 over several other isoenzymes (PRMT1, PRMT4 and PRMT6). Besides, C_4 demonstrated obvious cell apoptotic effect while reduced the cellular symmetric arginine dimethylation levels of SmD3 protein. The potency, small size, and synthetic accessibility of this compound class provide promising hit scaffold for medicinal chemists to further explore this series of PRMT5 inhibitors.

Interaction assessments of the first S-adenosylmethionine competitive inhibitor and the essential interacting partner methylosome protein 50 with protein arginine methyltransferase 5 by combined computational methods.

Protein arginine methyltransferase 5 (PRMT5) is the most promising anticancer target in PRMT family. In this study, based on the first S-adenosylmethionine (SAM) competitive small molecule inhibitor (17, compound number is from original paper) of PRMT5 reported in our recent paper, we determined the molecular mechanism of 17 interacting with PRMT5 by computational methods. Previously reported CMP5 was also thought of as a SAM competitive inhibitor of PRMT5, but the direct inhibition activity against PRMT5 at enzymatic level was not provided. Therefore, we tested the half-maximal inhibitory concentration (IC50) of CMP5 against PRMT5 at enzymatic level for the purpose of summarizing the interaction characteristics of SAM binding site inhibitors with PRMT5. Additionally, as the essential interacting partner of PRMT5, the binding attributes of the WD-repeat-containing protein MEP50 (methylosome protein 50) was investigated, and nine key residues that contribute most to PRMT5:MEP50 interaction were identified. These results could be helpful in discovering new potent and specific inhibitors of PRMT5, as well as in designing mutant residue assay to modulate the catalytic activity of PRMT5.

Biochemistry and regulation of the protein arginine methyltransferases (PRMTs).

Many key cellular processes can be regulated by the seemingly simple addition of one, or two, methyl groups to arginine residues by the nine known mammalian protein arginine methyltransferases (PRMTs). The impact that arginine methylation has on cellular well-being is highlighted by the ever growing evidence linking PRMT dysregulation to disease states, which has marked the PRMTs as prominent pharmacological targets. This review is meant to orient the reader with respect to the structural features of the PRMTs that account for catalytic activity, as well as provide a framework for understanding how these enzymes are regulated. An overview of what we understand about substrate recognition and binding is provided. Control of product specificity and enzyme processivity are introduced as necessary but flexible features of the PRMTs. Precise control of PRMT activity is a critical component to eukaryotic cell health, especially given that an arginine demethylase has not been identified. We therefore conclude the review with a comprehensive discussion of how protein arginine methylation is regulated.