title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.
abstract: Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.
authors.affiliation: Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.
authors.forename: Hideharu
authors.initials: H
authors.lastname: Hashimoto
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: June E
authors.initials: JE
authors.lastname: Pais
authors.affiliation: Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhang
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Lana
authors.initials: L
authors.lastname: Saleh
authors.affiliation: 1] Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia 30602, USA [2] Sector 22, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA.
authors.forename: Zheng-Qing
authors.initials: ZQ
authors.lastname: Fu
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Dai
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry / Q000378:metabolism* / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006860:Hydrogen Bonding; D051379:Mice; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009253:Naegleria / Q000201:enzymology* / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: C560974:5-carboxylcytosine; C560973:5-formylcytosine; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011518:Proto-Oncogene Proteins; C540839:TET1 protein, mouse; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C466427:AlkB protein, E coli