Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5-RNA complexes.

Autor:	Kaur S; Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China., Tam NY; Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China., McDonough MA; The Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK., Schofield CJ; The Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK., Aik WS; Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2022 Apr 22; Vol. 50 (7), pp. 4148-4160.
DOI:	10.1093/nar/gkac195
Abstrakt:	AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5'-3' orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139. (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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