Elucidation of the Mechanism for the Substrate Preference of TET Proteins

Abstract: Methylation of cytosine in DNA is considered as an important epigenetic marker， which takes part in various biological processes. Ten-eleven translocation (TET) proteins are key players involving in DNA demethylation through mediating the processive oxidation of 5-methylcytosine (5mC). It can convert 5mC to 5-hydroxymethylcytosine (5hmC)， then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). According to previous reports and our biochemical experiments， although TET2 could catalyze the oxidation of 5mC， 5hmC and 5fC， the catalytic efficiencies are different. The efficiency for 5mC is the highest while the efficiency for 5fC is the lowest. Biochemical experiments and crystal structures all showed TET2 could recognize and bind to its different substrates with similar binding affinities. Structure modelling and QM/MM calculations suggested different orientations of the substrates bases on the hydrogen abstraction reaction step may result in substrate preference.Further biochemical experiments such as Kinetic isotope effect (KIE) experiments and Stopped-flow spectroscopy experiments validated the hypothesis and indicated the difference in catalytic efficiencies indeed resulted from the differences in hydrogen abstraction rate. Our studies for the first time demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. In addition， it will light up the road for the development of small molecule regulators against TET proteins.

CSTR: 32200.14.cjcb.2016.01.0001