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Crystal structure of human ALKBH5 in complex with N-oxalylglycine (NOG) and m6A-containing ssRNACrystal structure of human ALKBH5 in complex with N-oxalylglycine (NOG) and m6A-containing ssRNA
Structural highlights
FunctionALKB5_HUMAN Dioxygenase that demethylates RNA by oxidative demethylation: specifically demethylates N(6)-methyladenosine (m(6)A) RNA, the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Requires molecular oxygen, alpha-ketoglutarate and iron. Demethylation of m(6)A mRNA affects mRNA processing and export and is required for spermatogenesis.[1] [2] Publication Abstract from PubMedAlkB 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. Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5-RNA complexes.,Kaur S, Tam NY, McDonough MA, Schofield CJ, Aik WS Nucleic Acids Res. 2022 Apr 22;50(7):4148-4160. doi: 10.1093/nar/gkac195. PMID:35333330[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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