How Human TET2 Enzyme Catalyzes the Oxidation of Unnatural Cytosine Modifications in Double-Stranded DNA

Methylation of cytosine bases is strongly linked to gene expression, imprinting, aging, and carcinogenesis. The teneleven translocation (TET) family of enzymes, which are Fe(II)/2species to dealkylate the lesioned bases to an unmodified cytosine. Recently, it has been shown that the TET2 enzyme can catalyze promiscuously DNA substrates containing an unnatural alkylated cytosine. Such unnatural substrates of TET can be used as direct probes for measuring the TET activity or capturing TET from cellular samples. Herein, we studied the catalytic mechanisms during the oxidation of the unnatural C5-position modifications (5ethylcytosine (5eC), 5-vinylcytosine (5vC), and 5-ethynylcytosine (5eyC)) and the demethylation of N4-methylated lesions (4methylcytosine (4mC) and 4,4-dimethylcytosine(4dmC)) of the cytosine base by the TET2 enzyme using molecular dynamics (MD) and combined quantum mechanics and molecular mechanics (QM/MM) computational approaches. The results reveal that the chemical nature of the alkylation of the double-stranded (ds) DNA substrates induces distinct changes in the interactions in the binding site, the second coordination sphere, and long-range correlated motions of the ES complexes. The rate-determining hydrogen atom transfer (HAT) is faster in N4-methyl substituent substrates than in the C5-alkylations. Importantly, the calculations show the preference of hydroxylation over desaturation in both 5eC and 5vC substrates. The studies elucidate the posthydroxylation rearrangements of the hydroxylated intermediates of 5eyC and 5vC to ketene and 5-formylmethylcytosine (5fmC), respectively, and hydrolysis of the hemiaminal intermediate of 4mC to formaldehyde and unmodified cytosine proceeds exclusively in an aqueous solution outside of the enzyme environment. Overall, the studies show that the chemical nature of the unnatural alkylated cytosine substrates exercises distinct effects on the binding interactions, reaction mechanism, and dynamics of TET2.

Automated summary

This study investigates the effects of unnatural alkylated cytosine substrates on the binding interactions, reaction mechanism, and dynamics of the TET2 enzyme. The results reveal that the chemical nature of the substrates induces changes in the binding site, coordination sphere, and long-range correlated motions of the enzyme-substrate complexes. The rate-determining hydrogen atom transfer is faster in N4-methylated substrates compared to C5-alkylated substrates. The use of unnatural substrates allows for the measurement of TET activity or the capture of TET from cellular samples.