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Enzymes of the radicalS-adenosyl-l-methionine (radical SAM, RS) superfamily, the largest in nature, catalyze remarkably diverse reactions initiated by H-atom abstraction. Glycyl radical enzyme activating enzymes (GRE-AEs) are a growing class of RS enzymes that generate the catalytically essential glycyl radical of GREs, which in turn catalyze essential reactions in anaerobic metabolism. Here, we probe the reaction of the GRE-AE pyruvate formate-lyase activating enzyme (PFL-AE) with the peptide substrate RVSG₇₃₄YAV, which mimics the site of glycyl radical formation on the native substrate, pyruvate formate-lyase. Time-resolved freeze-quench electron paramagnetic resonance spectroscopy shows that at short mixing times reduced PFL-AE + SAM reacts with RVSG₇₃₄YAV to form the central organometallic intermediate, Ω, in which the adenosyl 5′C is covalently bound to the unique iron of the [4Fe–4S] cluster. Freeze-trapping the reaction at longer times reveals the formation of the peptide G₇₃₄• glycyl radical product. Of central importance, freeze-quenching at intermediate times reveals that the conversion of Ω to peptide glycyl radical is not concerted. Instead, homolysis of the Ω Fe–C5′ bond generates the nominally “free” 5′-dAdo• radical, which is captured here by freeze-trapping. During cryoannealing at 77 K, the 5′-dAdo• directly abstracts an H-atom from the peptide to generate the G₇₃₄• peptide radical trapped in the PFL-AE active site. These observations reveal the 5′-dAdo• radical to be a well-defined intermediate, caught in the act of substrate H-atom abstraction, providing new insights into the mechanistic steps of radical initiation by RS enzymes. 

Abstract DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO.