The AidB Component of the Escherichia coli Adaptive Response to Alkylating Agents Is a Flavin-Containing, DNA-Binding Protein

Abstract

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.Two general classes of environmental and laboratory chemicals are known to alkylate DNA. S N 1 reagents (e.g., methylnitrosourea and N-methyl-NЈ-nitro-N-nitrosoguanidine [MNNG]) react primarily with the N 7 and O 6 positions of guanine, N 3 of adenine, O 6 or O 4 of pyrimidines, and the nonphosphodiester oxygen atoms of the phosphate backbone. In contrast, S N 2 agents (e.g., methyl methanesulfonate and dimethylsulfate) react primarily with the N 1 position of adenine and N 3 of cytosine (40). Microorganisms generate endogenous methylation compounds, such as S-adenosylmethionine, and are exposed to exogenous methylating substances that can modify their DNA, RNA, and other cellular components.To overcome the mutagenic and toxic effects of DNA methylation, Escherichia coli possesses a variety of DNA repair enzymes, some of which are induced as part of the "adaptive response to alkylating agents" (40,(43)(44)(45)(46). A key component of this process is the Ada protein, associated with three distinct activities. The amino-terminal domain of Ada catalyzes a methyl phosphotriester methyltransferase reaction that repairs methyl phosphotriesters in the DNA backbone while irreversibly methylating its Cys-38 side chain. Similarly, the carboxylterminal domain of Ada possesses 4-methyl-T and 6-methyl-G methyltransferase activities that irreversibly methylate Cys-321. In addition to the single-turnover reactions catalyzed by Ada, the protein (in its Cys-38-methylated form) functions as an activator that enhances transcription of its own gene as well as those encoding AlkA, AlkB, and AidB. AlkA is a 3-methyl-A DNA glycosylase that removes the alkylated base to create abasic sites in the DNA product. AlkB is an Fe(II)-and ␣-ketoglutarate-dependent hydroxylase that uses oxidative demethylation chemistry to reverse methylation damage to 1-methyl-A and 3-methyl-C (42). In contrast to the situation for Ada, AlkA, and AlkB, the role of AidB in the adaptive response process remains uncharacterized.The AidB sequence reveals a close relationship to acylcoenzyme A (CoA) dehydrogenases, ...

Structure and DNA binding of alkylation response protein AidB

“…At domain IV, pairs of ␣M and ␣O helices across the A/B dyad axis of symmetry create a highly electropositive concave groove ϳ20 Å wide, perfectly sized to accommodate a DNA duplex. In addition, the mouth of the conserved ACAD acyl-CoA substrate channel is positively charged, and proteolytic cleavage of AidB near this region (Met-194) was shown previously to be protected in the presence of DNA (7). To test these two regions as potential DNA binding sites, we measured the affinity of site-directed mutant proteins for 25mer dsDNA using a fluorescence anisotropy assay (Fig.…”

“…AidB has been shown recently to bind stoichiometric amounts of redox active FAD, and weak isovaleryl-CoA dehydrogenase (IVD) activity has been detected in vitro from both crude cell extracts and purified preparations (4,7). However, the visible spectrum of AidB's flavin was unaffected by isovaleryl-CoA, suggesting that fatty acyl-CoAs are not substrates for the enzyme (7).…”

“…However, the visible spectrum of AidB's flavin was unaffected by isovaleryl-CoA, suggesting that fatty acyl-CoAs are not substrates for the enzyme (7). Importantly, AidB was shown to bind dsDNA, and homology modeling predicted the DNA binding site to be located in the C-terminal region of the protein (7). The presence of a redoxactive flavin and DNA binding activity led to the suggestion that AidB might catalyze the direct repair of methylated DNA by a dehydrogenase mechanism (7).…”

“…AidB is related in sequence to acyl-CoA dehydrogenases (ACADs), which use a flavin adenine dinucleotide (FAD) to catalyze the ␣,␤-dehydrogenation of acyl-CoA conjugates (4,7). AidB has been shown recently to bind stoichiometric amounts of redox active FAD, and weak isovaleryl-CoA dehydrogenase (IVD) activity has been detected in vitro from both crude cell extracts and purified preparations (4,7).…”

Alleviation of 1, N 6 -ethanoadenine genotoxicity by the Escherichia coli adaptive response protein AlkB

“…AlkA, as mentioned above, is a 3-methyladenine DNA glycosylase responsible for excision of damaged bases as the first step in the base excision repair pathway. AidB is homologous to human isovaleryl CoA dehydrogenase, a central player in the leucine metabolism pathway, but its functional role in the adaptive response has not yet been elucidated beyond its ability to bind double-stranded DNA (9).…”