期刊
NUCLEIC ACIDS RESEARCH
卷 50, 期 7, 页码 4148-4160出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkac195
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资金
- Research Grants Council of Hong Kong Early Career Scheme 2019/20 [22301719]
- Hong Kong Baptist University Tier 2 Start-Up Grant [RC-SGT2/18-19/SCI/003]
- Wellcome Trust [106244/Z/14/Z]
- Hong Kong Baptist University
ALKBH5 is an enzyme responsible for demethylating RNA and has emerged as a potential target for cancer treatment. This study provides insight into the substrate recognition and demethylation mechanism of ALKBH5, and reveals its preference for specific sequences.
AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N-6-methyladenosine (m(6)A), 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 m(6)A 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 m(6)A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m(6)A-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)m(6)AC consensus sequence motif. The results support a mechanism involving formation of an m(6)A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.
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