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, ...