Structure and DNA binding of alkylation response protein AidB

Abstract

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 flavindependent 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-Å 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.acyl-CoA dehydrogenase ͉ adaptive response ͉ DNA repair ͉ flavin adenine dinucleotide

Specific DNA Binding and Regulation of Its Own Expression by the AidB Protein in Escherichia coli

“…As expected, the construct that expresses only the DNA binding domain (AidB I-III) showed no IVD activity. These findings clearly indicated that AidB I-III is responsible for its catalytic activity, as predicted by structural analyses (2).…”

“…Means and standard deviations have been calculated from four independent assays. residues, comprise the acyl-CoA dehydrogenase activity, and domain IV comprises the DNA binding activity (2). In order to determine whether the entire protein is required for the repressor activity or if the acyl-CoA dehydrogenase domain is dispensable, we constructed plasmids that express only the dehydrogenase domain or the DNA binding domain and tested them for AidB regulatory activity in vivo.…”

“…Human isovaleryl-CoA dehydrogenase exhibits a specific activity of 8.2 to 11.7 mol min Ϫ1 (mg protein) Ϫ1 (1,17), about 2 orders of magnitude higher than that of AidB. Recent structural studies revealed several unique features that distinguish AidB's FAD cavity from the ACAD active sites despite the conservation of their general properties (2). These observations provided a rationale for AidB's limited acyl-CoA dehydrogenase activity (20), suggesting that fatty acyl-CoAs are too large to fit the AidB active site and cannot in fact be the main substrates of this enzyme.…”

“…AidB has been reported to show weak isovaleryl-CoA dehydrogenase (IVD) activity (12,20) and to exhibit nonspecific DNA binding activity (20). The crystal structure of AidB reveals a unique quaternary organization, a peculiar FAD active site that provides a rationale for its limited dehydrogenase activity, and a putative DNA binding site located in the C-terminal region of the protein (2). aidB expression is regulated by several different mechanisms.…”

Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase fromCaenorhabditis elegans

“…Molecular replacement was used to determine the crystal structure of cVLCAD using the structure of E. coli AidB (PDB code 3djl; Bowles et al, 2008), which has 30% sequence identity to cVLCAD, as a search model. One unique solution with two monomers was obtained with a likelihood score of 310.8 and a Z score of 16.7 using the program Phaser (McCoy et al, 2007;Collaborative Computational Project, 1994).…”