Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes
AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA <i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mas...
| Main Authors: | , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Oxford University Press
2022
|
| _version_ | 1797110922867638272 |
|---|---|
| author | Kaur, S Tam, NY McDonough, MA Schofield, CJ Aik, WS |
| author_facet | Kaur, S Tam, NY McDonough, MA Schofield, CJ Aik, WS |
| author_sort | Kaur, S |
| collection | OXFORD |
| description | AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA <i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>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<sup>6</sup>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<sup>6</sup>A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m<sup>6</sup>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<sup>6</sup>AC consensus sequence motif. The results support a mechanism involving formation of an m<sup>6</sup>A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139. |
| first_indexed | 2024-03-07T08:01:33Z |
| format | Journal article |
| id | oxford-uuid:14cb2061-c2cf-4529-8446-50b2201bdb88 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T08:01:33Z |
| publishDate | 2022 |
| publisher | Oxford University Press |
| record_format | dspace |
| spelling | oxford-uuid:14cb2061-c2cf-4529-8446-50b2201bdb882023-10-06T11:20:11ZMechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexesJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:14cb2061-c2cf-4529-8446-50b2201bdb88EnglishSymplectic ElementsOxford University Press2022Kaur, STam, NYMcDonough, MASchofield, CJAik, WSAlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA <i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>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<sup>6</sup>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<sup>6</sup>A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m<sup>6</sup>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<sup>6</sup>AC consensus sequence motif. The results support a mechanism involving formation of an m<sup>6</sup>A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139. |
| spellingShingle | Kaur, S Tam, NY McDonough, MA Schofield, CJ Aik, WS Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes |
| title | Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes |
| title_full | Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes |
| title_fullStr | Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes |
| title_full_unstemmed | Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes |
| title_short | Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes |
| title_sort | mechanisms of substrate recognition and n6 methyladenosine demethylation revealed by crystal structures of alkbh5 rna complexes |
| work_keys_str_mv | AT kaurs mechanismsofsubstraterecognitionandn6methyladenosinedemethylationrevealedbycrystalstructuresofalkbh5rnacomplexes AT tamny mechanismsofsubstraterecognitionandn6methyladenosinedemethylationrevealedbycrystalstructuresofalkbh5rnacomplexes AT mcdonoughma mechanismsofsubstraterecognitionandn6methyladenosinedemethylationrevealedbycrystalstructuresofalkbh5rnacomplexes AT schofieldcj mechanismsofsubstraterecognitionandn6methyladenosinedemethylationrevealedbycrystalstructuresofalkbh5rnacomplexes AT aikws mechanismsofsubstraterecognitionandn6methyladenosinedemethylationrevealedbycrystalstructuresofalkbh5rnacomplexes |