Catalytic Space Engineering as a Strategy to Activate C–H Oxidation on 5-Methylcytosine in Mammalian Genome

Abstract

Conditional remodeling of enzyme catalysis is a formidable challenge in protein engineering. Herein, we have undertaken a unique active site engineering tactic to command catalytic outcomes. With ten−eleven translocation (TET) enzyme as a paradigm, we show that variants with an expanded active site significantly enhance multistep C−H oxidation in 5-methylcytosine (5mC), whereas a crowded cavity leads to a single-step catalytic apparatus. We further identify an evolutionarily conserved residue in the TET family with a remarkable catalysis-directing ability. The activating variant demonstrated its prowess to oxidize 5mC in chromosomal DNA for potentiating expression of genes including tumor suppressors.

The pH‐Dependence of the Hydration of 5‐Formylcytosine: an Experimental and Theoretical Study

“…Despite these seemingly low values, the aldehyde hydrate forms may nevertheless play an essential role in oxidation reactions to the respective 5-carboxy derivatives in a way well established for aldehyde oxidations mediated by chemical oxidants or dehydrogenase enzymes. [38,[54][55][56] In both areas evidence for the stabilization of hydrate intermediates through directed hydrogen bonding interactions has been found, which is in full support of the gas phase calculations with explicit water molecules in the current study. This may also provide a rational basis for the proposed high abundance of 5-formylcytosine hydrates reported by Burrows et al in base-flipping kinetics studies.…”

“…This may also provide a rational basis for the proposed high abundance of 5-formylcytosine hydrates reported by Burrows et al in base-flipping kinetics studies. [36] A potential TET-mediated oxidation of fC through the respective hydrate [38] moves this process mechanistically closer to that of hmC, where recent theoretical studies have established similar reaction barriers for initial OÀ H vs. CÀ H hydrogen abstraction steps. [19,57] Experimental Section Energy of hydration ΔG hyd : The reaction of aldehydes (A) with water in aqueous solution yields the respective hydrate A_hyd according to Eq.…”

“…In a recent NMR study on the melting kinetics of 5‐formylcytosine in dsDNA no evidence for the respective geminal diol form was found in 1 H or 13 C NMR measurements [37] . This does, of course, not exclude transient hydrate formation in TET2‐mediated fC oxidation reactions [38] . Direct measurements of the hydration equilibrium of fC have not yet been reported.…”

Computational Investigations of Selected Enzymes From Two Iron and α-ketoglutarate-Dependent Families

“…Sappa et al proposed that if the active site were altered, a more efficient turnover of the 5mC analogues could be found. [110] The group began by replacing a variety of hydrophobic and polar residues with alanine that were near to the active site. From there, the enzyme was exposed to 5mC and the concentrations of the intermediates (5hmC, 5fC and 5caC) were measured.…”

“…The authors believe the active site of TET2 may be crowded and smaller side chains may aid in a higher catalytic efficiency of TET2. [110] E. Substrate preference While a majority of the work done this far on the TET2 enzyme has been with respect to DNA substrates, it is worth discussing the ability of TET enzymes to repair RNA methylated bases. The various forms of methylated cytosine such as 5mC, 5hmC, 5fC and 5caC can also be found in different types of RNA (mRNA, tRNA, rRNA and ncRNA).…”