Conformational flexibility influences structure–function relationships in nucleic acid<i>N</i>-methyl demethylases

Waheed, Sodiq O.; Ramanan, Rajeev; Chaturvedi, Shobhit S.; Ainsley, Jon; Evison, Martin; Ames, Jennifer M.; Schofield, Christopher J.; Christov, Christo; Karabencheva‐Christova, Tatyana G.

doi:10.1039/c9ob00162j

Cited by 18 publications

(17 citation statements)

References 62 publications

(84 reference statements)

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“…Details of the mechanism leading to the ferryl intermediate and studies on model systems have been reported [15– 26a] . Following substrate binding, the consensus 2OG oxygenase mechanism [7,12– 26a–c] involves initial generation of a Fe(III)‐superoxo complex; oxidative decarboxylation of 2OG generates a Fe(II)‐peroxysuccinate intermediate which reacts to give a high‐spin (S=2) state ferryl intermediate, which abstracts a hydrogen atom from the substrate. Hydrogen atom transfer (HAT) through a high spin surface is favored by spin exchange enhanced reactivity at the transition state, [27,28a] generating an Fe(III)‐hydroxyl intermediate that undergoes a low barrier hydroxyl rebound reaction with the substrate radical carbon to complete hydroxylation [Scheme 1].…”

Section: Introductionmentioning

confidence: 99%

What Is the Catalytic Mechanism of Enzymatic Histone N‐Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field?

Ramanan

Waheed

Schofield

et al. 2021

Chemistry A European J

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Arginine methylation is an important mechanism of epigenetic regulation. Some Fe(II) and 2‐oxoglutarate dependent Jumonji‐C (JmjC) Nϵ‐methyl lysine histone demethylases also have N‐methyl arginine demethylase activity. We report combined molecular dynamic (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) studies on the mechanism of N‐methyl arginine demethylation by human KDM4E and compare the results with those reported for N‐methyl lysine demethylation by KDM4A. At the KDM4E active site, Glu191, Asn291, and Ser197 form a conserved scaffold that restricts substrate dynamics; substrate binding is also mediated by an out of active site hydrogen‐bond between the substrate Ser1 and Tyr178. The calculations imply that in either C−H or N−H potential bond cleaving pathways for hydrogen atom transfer (HAT) during N‐methyl arginine demethylation, electron transfer occurs via a σ‐channel; the transition state for the N−H pathway is ∼10 kcal/mol higher than for the C−H pathway due to the higher bond dissociation energy of the N−H bond. The results of applying external electric fields (EEFs) reveal EEFs with positive field strengths parallel to the Fe=O bond have a significant barrier‐lowering effect on the C−H pathway, by contrast, such EEFs inhibit the N−H activation rate. The overall results imply that KDM4 catalyzed N‐methyl arginine demethylation and N‐methyl lysine demethylation occur via similar C−H abstraction and rebound mechanisms leading to methyl group hydroxylation, though there are differences in the interactions leading to productive binding of intermediates.

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Section: Introductionmentioning

confidence: 99%

What Is the Catalytic Mechanism of Enzymatic Histone N‐Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field?

Ramanan

Waheed

Schofield

et al. 2021

Chemistry A European J

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“…Because the conformational dynamics play an important role in the catalysis by the AlkB dioxygenase [15][16][17][18], it would be interesting to correlate specific conformational transitions of the enzyme-substrate complex with the individual steps of the process. Earlier, we applied the stopped-flow (SF) kinetic analysis of conformational dynamics to several repair DNA glycosylases and apurinic/apyrimidinic endonucleases as well as their DNA substrates to demonstrate how conformational rearrangements induce and follow the recognition, binding, and chemical events [19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB

2019

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AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive 'oxyferryl' enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single-or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N 1 -Methyladenine (m 1 A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein's Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye-quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction.

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“…When co-expressed as separate polypeptides in E. coli , these domains formed a stable complex [ 18 ]. Additionally, computational studies of Waheed and coworkers have revealed that the N- and C-terminal domains move with respect to one another in a manner likely important for substrate binding [ 44 ]. Therefore, testing the influence of the dimerization on activity by mutating amino acids in the C-terminal domain would be questionable.…”

Section: Discussionmentioning

confidence: 99%

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

Marcinkowski

Pilžys

Garbicz

et al. 2021

IJMS

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The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

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Conformational flexibility influences structure–function relationships in nucleic acidN-methyl demethylases

Abstract: Distinct conformational effects influence structure-function correlations in AlkB and FTO.

Cited by 18 publications

References 62 publications

What Is the Catalytic Mechanism of Enzymatic Histone N‐Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field?

What Is the Catalytic Mechanism of Enzymatic Histone N‐Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field?

A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

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