Download presentation
Presentation is loading. Please wait.
Published by雁 鲁 Modified over 7 years ago
1
Structure of the hABH3 DNA demethylase implicates an oxidized leucine residue in catalysis
2
Background Cells are exposed to alkylating agents produced both endogeneously and in the environment Such agents may attack the O- and C-atoms of nucleobases. Unless repaired, the lesions may be mutagenic and/or cytotoxic N O H C 3 R 2 1 4 5 6 T G 9 8 7 A Mutagenic Non-mutagenic/cytotoxic
3
(3-alkA, 7-alkA, 3-alkG, 7-alkG)
DNA RNA N-alkylation 1-alkA 3-alkA 7-alkA 3-alkG 7-alkG 3-alkT/U 3-alkC AlkB-homologues (1-meA, 3meC, others?) O-alkylation O6-alkG O4-alkT O2-alkT O2-alkC Cytotoxicity mutations AGT (O6-alkG >>O4-alkT) NER (O4-alkT) BER (MPG) (3-alkA, 7-alkA, 3-alkG, 7-alkG) Alk. agent Mutations cytotoxicity Several DNA-repair pathways contribute to the reversal of alkylation damage Of these, the oxidative demethylation by the AlkB-family of enzymes is the most recently discovered N/alkyleringer er generelt mindre mutagene enn O alkyleringer, men er cytotoksiske
4
AlkB One of four proteins involved in the adaptive response to DNA alkylation damage in E.Coli Belong to a superfamily of 2-oxoglutarate- and iron-dependent oxygenases Highly conserved from bacteria to humans Mediate direct reversal of alkylation damage using a novel iron-catalyzed oxidative demethylation mechanism Activity against DNA and RNA 8 human homologues, hABH1-8, have been identified. Two of these, hABH 2 and 3 have been shown to have oxidative demethylase activity
5
Aims A high degree of sequence homology is observed between AlkB and the hABH2/3 proteins. Eight of the strands (7-13) form a jelly roll fold
6
Oxidative demethylation - mechanism
RO OR N H 2 O C A NH2 2 C H 3 H 2 N N + C H 2 N N + 1-meA N N N N AlkB, hABH2/3 N O O RO OR RO OR Fe2+ NH2 N O H3C O CO O H NH2 2 2 + + H2C + H O O H + N O O O O N 3-meC O O O O H O H O succinate RO OR RO 2-oxo-glutarate OR
7
Crystallization of hABH3
Aims As for many other DNA-repair proteins, crystallization of the full-length protein was not successful This may be due to the presence of unstructured regions in the protein Deletion of the 69 N-terminal residues of hABH3 yielded, however, crystals in complex with iron and the co-substrate 2-OG that diffrected to 1.5Å Eight of the strands (7-13) form a jelly roll fold
8
Aims Overall fold of hABH3
hABH3 forms a jelly roll fold consisting of two -sheets characteristic of many metal-binding and non-metal-binding proteins. 4 and 5 form a unique hairpin motif that creates a lid over one side of the jelly roll barrel Eight of the strands (7-14) form a jelly roll fold. alpha 2 to beta 14 conserved among the dioxygenases
9
The electron density maps reveals additional density at Leu177
The electron density maps revealed a surprising density at Leu 177 (arrow). The shape and direction of this density indicated that is was caused by a hydroxyl- or a carbonyl group at this residue
10
Leu-carbonylation is supported by MS data
Aims Trypsin digestion and MALDI-TOF peptide fingerptinting revealed a modified form of the Leu177-containing fragment corresponding to a carbonylated form (14 kDa shift). This form is not observed in the mutant Leu177Ala Eight of the strands (7-13) form a jelly roll fold
11
Mutations in Iron-binding residues
Aims Close-up view of the iron coordination. H191, D193, and H257 bind at one face of the single iron atom (coloured violet). 2OG chelates the iron in a bi-dentate manner with the 1-carboxyl group opposite H257 and the keto group opposite the acidic residue. Similar “off-line” 2OG coordination13 is observed in CarC30 and ANS14. A water molecule (coloured red) occupies the sixth position in the distorted octahedral coordination of the iron atom.
12
Mutations in 2-OG-binding residues 1
Aims Eight of the strands (7-13) form a jelly roll fold
13
Mutations in 2-OG-binding residues 2
Aims Eight of the strands (7-13) form a jelly roll fold
14
A potential role for Ald-Leu in catalysis?
Aims A carbonylated leucine has previously not been associated with any catalytic mechanism Thus: Is the modification a prerequisite for catalysis, or is it formed as a side-effect caused by uncoupled decarboxylation? Har sjekket mutanter med nedsatt kat aktivitet. Disse har I de fleste tilfeller sterkt nedsatt karbonylering, men ikke entydelige resultat. Ser ut til at mutatner I residier viktige for substratbinding, fremdeles har karbonylering, mens mutanter I residier viktige for dekarboksylaseaktivitet, har nedsatt karbonylering (eks – jernbindende residier.. Prøver å skille modifisert/umodifisert form for å se på katalytisk aktivitet (ARP) Karbonylere alt ved å tilsette Fe/2og og deretter kjøre assay for demetylaseaktivitet Mutere leu til gln og glu-mutasjoner for å se om aktiviteten endres
15
Aims Hypothetic mechanism
Viktig: Nevn at metyl må være der for å få katalyse, pga direkte binding til L177 ald og displacement va 2OG. Med feil substrat går ikke 2OG online, The 2OG is in “off-line” coordination to the iron atom in the absence of substrate, as observed in the crystal structure (presented as a ball-and-stick cartoon to the left). (II) Binding of substrate most likely causes the 2OG to change to “in-line” coordination at the expense of the iron-bound water molecule and conformational change of L177 and R275, as illustrated in the model of the substrate docked into the active site (left). The 1-meA substrate was manually docked into the active site of hABH3 using the enzyme-substrate complex of the Fe(II)/2OG-dependent dioxygenase FIH and HIF C-terminal peptide (PDBid 1H2L) to position the 1-meA base close to the Fe atom. The MGIH motif was too close to the jelly roll core to accommodate either 1-meA/3meC-containing ssDNA or dsDNA, either as a result of natural close conformation in the unbound state or of close contacts with a symmetry mate in the crystal lattice. The open coordination site, directed towards the substrate, could then accommodate the molecular oxygen, oriented by the help of R275. Oxygen reacts with 2OG in a mechanism that is not fully understood, in which 2OG is transformed to a carbonate-succinate mixed-anhydride bound to an Fe(III)-hydroxyl radical or Fe(IV)-oxo intermediate. (III) The activated oxygen species abstract a hydrogen from the 1-methyl group and forms a substrate radical plus an Fe(III)-hydroxide intermediate. (IV) Recombination of the hydroxyl group and the radical forms the hydroxylated substrate. The Fe(II)-transition state is restored, the carbonate-succinate mixed-anhydride is released as CO2 and succinate. 1-hydroxymethyladenine and 3-hydroxymethylcytosine are not stable compounds and will spontaneously yield formaldehyde and repaired adenine and cytosine bases.
16
People involved in the present work
NTNU, IKM Ottar Sundheim Mirta de Sousa Cathrine B. Vågbø Vivi Talstad Per Arne Aas Emadoldin Feyzi Finn Drabløs Hans Krokan Geir Slupphaug UiO Magnar Bjørås TSRI John A. Tainer
Similar presentations
© 2025 SlidePlayer.com Inc.
All rights reserved.