Complementary Steric Engineering at the Protein-Ligand Interface for Analogue-Sensitive TET Oxygenases

Journal of the American Chemical Society
Babu SudhamallaKabirul Islam

Abstract

Ten-eleven translocation (TET) enzymes employ O2, earth-abundant iron, and 2-ketoglutarate (2KG) to perform iterative C-H oxidation of 5-methylcytosine in DNA to control expression of the mammalian genome. Given that more than 60 such C-H oxygenases are present in humans, determining context-dependent functions of each of these enzymes is a pivotal challenge. In an effort to tackle the problem, we developed analogue-sensitive TET enzymes to perturb the activity of a specific member. We rationally engineered the TET2-2KG interface to develop TET2 variants with an expanded active site that can be specifically inhibited by the N-oxalylglycine (NOG) derivatives carrying a complementary steric "bump". Herein, we describe the identification and engineering of a bulky gatekeeper residue for TET proteins, characterize the orthogonal mutant-inhibitor pairs, and show generality of the approach. Employing cell-permeable NOG analogues, we show that the TET2 mutant can be specifically inhibited to conditionally modulate cytosine methylation in chromosomal DNA in intact human cells. Finally, we demonstrate application of the orthogonal mutant-inhibitor pair to probe transcriptional activity of a specific TET member in cells. Our work provide...Continue Reading

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Citations

Jan 9, 2019·Chembiochem : a European Journal of Chemical Biology·Shana WagnerKabirul Islam
Aug 14, 2020·Chembiochem : a European Journal of Chemical Biology·Jenna N BeyerGeorge M Burslem
Apr 25, 2021·Bioorganic & Medicinal Chemistry·Anand D TiwariJames G Phillips
Jul 30, 2021·Journal of the American Chemical Society·Sushma SappaKabirul Islam
Apr 15, 2020·Journal of the American Chemical Society·Shubhendu PaleiDaniel Summerer
Sep 9, 2021·ACS Chemical Biology·Valerie ScottKabirul Islam

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