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Electrochemical characterization of Escherichia coli adaptive response protein AidB.

ABSTRACT: When exposed to known DNA-damaging alkylating agents, Escherichia coli cells increase production of four DNA repair enzymes: Ada, AlkA, AlkB, and AidB. The role of three enzymes (Ada, AlkA, and AlkB) in repairing DNA lesions has been well characterized, while the function of AidB is poorly understood. AidB has a distinct cofactor that is potentially related to the elusive role of AidB in adaptive response: a redox active flavin adenine dinucleotide (FAD). In this study, we report the thermodynamic redox properties of the AidB flavin for the first time, both for free protein and in the presence of potential substrates. We find that the midpoint reduction potential of the AidB flavin is within a biologically relevant window for redox chemistry at -181 mV, that AidB significantly stabilizes the flavin semiquinone, and that small molecule binding perturbs the observed reduction potential. Our electrochemical results combined with structural analysis allow for fresh comparisons between AidB and the homologous acyl-coenzyme A dehydrogenase (ACAD) family of enzymes. AidB exhibits several discrepancies from ACADs that suggest a novel catalytic mechanism distinct from that of the ACAD family enzymes.

SUBMITTER: Hamill MJ

PROVIDER: S-EPMC3546729 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Flavin-induced oligomerization in Escherichia coli adaptive response protein AidB.

Project description:The process known as "adaptive response" allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 ± 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 Å resolution crystal structure in space group P3(2) that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.

| S-EPMC3217306 | biostudies-literature

Structure and transcriptional regulation of the Escherichia coli adaptive response gene aidB.

Project description:Expression of the Escherichia coli aidB gene is induced in vivo by alkylation damage in an ada-dependent pathway and by anaerobiosis or by acetate at pH 6.5 in an ada-independent fashion. In this report, we present data on aidB gene structure, function, and regulation. The aidB gene encodes a protein of ca. 60 kDa that is homologous to several mammalian acyl coenzyme A dehydrogenases. Accordingly, crude extracts from an aidB-overexpressing strain showed isovaleryl coenzyme A dehydrogenase activity. aidB overexpression also reduced N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis. Both ada- and acetate/pH-dependent induction of aidB are regulated at the transcriptional level, and the same transcriptional start point is used for both kinds of induction. Ada protein plays a direct role in aidB regulation: methylated Ada is able to bind to the aidB promoter region and to activate transcription from aidB in an in vitro transcription-translation system using crude E. coli extracts.

| S-EPMC197013 | biostudies-other

The AidB component of the Escherichia coli adaptive response to alkylating agents is a flavin-containing, DNA-binding protein.

Project description:Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. In order to begin to identify the role of AidB in the cell, the protein was purified to homogeneity, shown to possess stoichiometric amounts of flavin adenine dinucleotide (FAD), and confirmed to have low levels of isovaleryl-coenzyme A (CoA) dehydrogenase activity. A homology model of an AidB homodimer was constructed based on the structure of a four-domain acyl-CoA oxidase. The predicted structure revealed a positively charged groove connecting the two active sites and a second canyon of positive charges in the C-terminal domain, both of which could potentially bind DNA. Three approaches were used to confirm that AidB binds to double-stranded DNA. On the basis of its ability to bind DNA and its possession of a redox-active flavin, AidB is predicted to catalyze the direct repair of alkylated DNA.

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Structure and DNA binding of alkylation response protein AidB.

Project description:Exposure of Escherichia coli to alkylating agents activates expression of AidB in addition to DNA repair proteins Ada, AlkA, and AlkB. AidB was recently shown to possess a flavin adenine dinucleotide (FAD) cofactor and to bind to dsDNA, implicating it as a flavin-dependent DNA repair enzyme. However, the molecular mechanism by which AidB acts to reduce the mutagenic effects of specific DNA alkylators is unknown. We present a 1.7-A crystal structure of AidB, which bears superficial resemblance to the acyl-CoA dehydrogenase superfamily of flavoproteins. The structure reveals a unique quaternary organization and a distinctive FAD active site that provides a rationale for AidB's limited dehydrogenase activity. A highly electropositive C-terminal domain not present in structural homologs was identified by mutational analysis as the DNA binding site. Structural analysis of the DNA and FAD binding sites provides evidence against AidB-catalyzed DNA repair and supports a model in which AidB acts to prevent alkylation damage by protecting DNA and destroying alkylating agents that have yet to reach their DNA target.

| S-EPMC2563087 | biostudies-literature

A Quantitative Model Explains Single-Cell Dynamics of the Adaptive Response in Escherichia coli.

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Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli.

Project description:While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy.

| S-EPMC5429094 | biostudies-literature

Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB.

Project description:The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and ?-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N(6) atom of adenine. The observation of oxidation of a carbon at N(6) in EA prompted us to evaluate N(6)-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo.

| S-EPMC3363417 | biostudies-literature

Adaptive Genetic Robustness of Escherichia coli Metabolic Fluxes.

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Purification and Characterization of Glutathione Binding Protein GsiB from Escherichia coli.

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