The AidB protein belongs to the adaptive response to alkylating agents (25
), which is defined by the Ada regulon. Upon exposure to alkylating agents, the Ada protein transfers methyl groups from damaged DNA to itself. Once methylated, Ada becomes able to activate the expression of the ada
, and aidB
genes, which confer cellular resistance to the mutagenic and cytotoxic effects of these agents (7
While the biological roles of Ada, AlkA, and AlkB have been defined (21
), little is known of the biological function of AidB in vivo
. AidB was reported to possess weak isovaleryl-coenzyme A dehydrogenase activity (12
) and to be a nonspecific DNA binding protein (20
). The ability to bind DNA both specifically and nonspecifically is a similarity to the Ada protein, which binds DNA nonspecifically in its unmethylated form and becomes able to recognize specific target sequences upon self-methylation (23
). However, in the case of AidB protein, there is no indication that an interaction with damage or a damaging agent is required for the transition between specific and nonspecific binding. Instead, this is an intrinsic property of the unmodified AidB protein, since protein isolated from untreated cells is capable of both specific and nonspecific DNA binding (Fig. ) (20
). In this respect, the sequence specificity of AidB binding to DNA deserves some comments. Results presented in this paper demonstrate that AidB is able to regulate expression of its own promoter but not the ada
promoter, which also contains an A/T-rich region. Further investigations are needed to fully clarify AidB binding specificity, including functional experiments with other promoters containing or not containing UP elements. Furthermore, chromatin immunoprecipitation (ChIP)-chip experiments might also be designed in order to determine other potential regulatory targets.
Induction of a regulatory element that counteracts induction appears to be a common feature of other stress responses. When E. coli
cells are stressed by heat to cause induction of heat shock genes, the normal sigma factor, sigma 70, is induced. It has been suggested that this increased level of sigma 70 may compete with sigma 32 for RNA polymerase binding (5
), thereby contributing to the characteristic heat shock gene expression pattern in which a strong, immediate induction of heat shock genes is followed by a dampening of their expression, reducing expression to an intermediate level (6
). Induction of the SOS response results in induction of lexA
, which encodes the LexA repressor of SOS genes. Since LexA is cleaved and inactivated by an activated form of the RecA protein, it is thought that the elevated levels of the LexA protein result in a rapid restoration of repression as soon as LexA protein production exceeds its cleavage, thereby allowing intact, functional LexA to accumulate (14
The biological relevance of AidB autorepression is not as universal as the repression functions triggered as part of either heat shock or the SOS response, since it affects only aidB gene expression. In the absence of alkylation, this autorepression clearly functions to further reduce the level of expression (Fig. ). The need for cells to regulate the basal expression of AidB by the additional autoregulatory mechanism suggests that expression of AidB may have deleterious consequences that will require it to be kept at a minimum in normal, actively growing cells. The determination of why cells require such a complex series of regulatory mechanisms to control aidB expression will clearly require a determination of its exact function in response to alkylation damage.
Finally, we investigated the domain architecture of AidB by expressing and functionally characterizing the N- and C-terminal domains of the protein containing domains I to III and domain IV, respectively. The N-terminal region encompassing the first 439 residues (AidB I-III) was shown to exhibit the same levels of isovaleryl-CoA dehydrogenase (IVD) activity as the full-length protein.
However, the level of IVD activity observed in AidB is quite low compared to that in other acyl-CoA dehydrogenases. Human isovaleryl-CoA dehydrogenase exhibits a specific activity of 8.2 to 11.7 μmol min−1
), 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. The crystal structural analysis of AidB also identified a putative DNA binding site located in its C-terminal region. We found that the C-terminal domain alone (aa 440 to 541) (AidB IV) possesses DNA binding activity and can negatively regulate the expression of the aidB
gene at the same level as the full-length protein.
Thus, acyl-CoA dehydrogenase activity does not appear to be involved in modulation of the DNA binding properties of the AidB protein. It is possible, however, that DNA binding might affect the enzymatic activity of the AidB protein, allowing AidB to carry out repair activity. Although this event might need a large conformational change according to the three-dimensional structure of the protein, a similar mechanism of action has been described for the AlkB protein and for its human homologues ABH2 and ABH3. These proteins are able to repair methylated bases by oxidative demethylation upon DNA binding.