Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates
| Autor: | Kevin J. Bruemmer, Eva J Ge, Christopher J. Chang, Joel D.W. Toh, Diana A. Iovan, Dan He, Steven W. M. Crossley |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Models Molecular Oxygenase Protein Conformation Mutant 01 natural sciences chemistry.chemical_compound Models Cellular localization chemistry.chemical_classification Multidisciplinary biology Fatty Acids AlkB Homolog 5 RNA Demethylase RNA Demethylase Biochemistry Physical Sciences MCF-7 Cells Single-Cell Analysis Oxidation-Reduction one-carbon cycle 1.1 Normal biological development and functioning Iron AlkB Alpha-Ketoglutarate-Dependent Dioxygenase FTO Oxidative phosphorylation 010402 general chemistry RNA epigenetics 03 medical and health sciences Underpinning research Humans Epigenetics AlkB Homolog 5 Demethylation nonheme iron enzyme Base Sequence RNA Molecular m6A 0104 chemical sciences 030104 developmental biology Enzyme HEK293 Cells chemistry biology.protein formaldehyde Generic health relevance N6-Methyladenosine |
| Zdroj: | Proc Natl Acad Sci U S A Proceedings of the National Academy of Sciences of the United States of America, vol 117, iss 41 |
| Popis: | The AlkB family of non-heme-Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6 methyladenosine (m6A) but furnish different major products, N6 hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a directm6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for non-redundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.Significance StatementNon-heme iron enzymes FTO and ALKBH5 play central roles in epigenetic RNA regulation by catalyzing the oxidation of N6-methyladenosine (m6A) to produce N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here, we provide a mechanistic rationale for these distinct biochemical outcomes by identifying that ALKBH5 performs m6A demethylation via an unprecedented covalent-based mechanism with concomitant and rapid release of A and formaldehyde (FA), whereas FTO liberates hm6A to release A and FA over longer timescales. This work reveals foundational biochemical differences between these closely related but non-redundant epigenetic enzymes and identifies ALKBH5 as an endogenous source of rapid formaldehyde generation in cells. |
| Databáze: | OpenAIRE |
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