Dataset Information

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

ABSTRACT: AlkB is a Fe(II)/?-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 (?-ketoglutarate, ?KG) 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. N1-Methyladenine (m1A) 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.

SUBMITTER: Kanazhevskaya LY

PROVIDER: S-EPMC6943663 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Kanazhevskaya Lyubov Yu LY Alekseeva Irina V IV Fedorova Olga S OS

Molecules (Basel, Switzerland) 20191213 24

AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in <i>Escherichia coli</i>. 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 an ...[more]

PMID: 31847292

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Project description:3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 ?M and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k'cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.

Dataset Information

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

Publications

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

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