{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T18:57:57Z","timestamp":1792090677063,"version":"4.4.0"},"reference-count":49,"publisher":"Oxford University Press (OUP)","issue":"7","license":[{"start":{"date-parts":[[2022,3,25]],"date-time":"2022-03-25T00:00:00Z","timestamp":1648166400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,4,22]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>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\u2032-3\u2032 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\u00a0mechanism involving\u00a0formation of an m6A hemiaminal intermediate, followed by\u00a0efficient ALKBH5 catalyzed demethylation,\u00a0enabled by a proton shuttle network involving Lys132 and Tyr139.<\/jats:p>","DOI":"10.1093\/nar\/gkac195","type":"journal-article","created":{"date-parts":[[2022,3,14]],"date-time":"2022-03-14T16:24:05Z","timestamp":1647275045000},"page":"4148-4160","source":"Crossref","is-referenced-by-count":76,"title":["Mechanisms of substrate recognition and\n                    <i>N<\/i>\n                    6-methyladenosine demethylation revealed by crystal structures of ALKBH5\u2013RNA complexes"],"prefix":"10.1093","volume":"50","author":[{"given":"Simranjeet","family":"Kaur","sequence":"first","affiliation":[{"name":"Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nok Yin","family":"Tam","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4664-6942","authenticated-orcid":false,"given":"Michael A","family":"McDonough","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0290-6565","authenticated-orcid":false,"given":"Christopher J","family":"Schofield","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4414-173X","authenticated-orcid":false,"given":"Wei\u00a0Shen","family":"Aik","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2022,3,25]]},"reference":[{"key":"2022042200073177100_B1","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1038\/s41422-018-0040-8","article-title":"Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism","volume":"28","author":"Yang","year":"2018","journal-title":"Cell Res."},{"key":"2022042200073177100_B2","doi-asserted-by":"crossref","first-page":"e16210","DOI":"10.1371\/journal.pone.0016210","article-title":"Human AlkB homologue 5 is a nuclear 2-oxoglutarate dependent oxygenase and a direct target of hypoxia-inducible factor 1\u03b1 (HIF-1\u03b1)","volume":"6","author":"Thalhammer","year":"2011","journal-title":"PLoS One"},{"key":"2022042200073177100_B3","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.molcel.2012.10.015","article-title":"ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility","volume":"49","author":"Zheng","year":"2013","journal-title":"Mol. 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