Structure and function of BenM, a transcriptional activator from Acinetobacter sp. Strain ADP1

Abstract

BenM and CatM, members of the LysR-family of transcriptional regulators, activate the expression of the ben and cat genes needed for benzoate degradation by the bacterium Acinetobacter sp. Strain ADP1. These two proteins, which are similar in sequence and function, regulate more than a dozen chromosomal genes organized in multiple operons. Both BenM and CatM respond to the metabolite cis,cis-muconate. BenM, but not CatM, additionally responds to the effector benzoate. As described in this dissertation, BenM and CatM were purified to homogeneity and shown to regulate the expression of two genes, benP and benK, that form an operon adjacent to the benM gene. BenP and BenK, a putative outer membrane porin and an inner membrane permease, respectively, were predicted to contribute to aromatic compound catabolism in ADP1. The BenM and CatM regulators were equally important in benPK expression. Futhermore, although BenM activates the benABCDE genes in response to benzoate, only cis,cis-muconate was able to increase benPK expression. To characterize the interactions of BenM with the two distinct effectors, tryptophan fluorescence methods were used. BenM was able to bind to benzoate and to cis,cis-muconate, and the affinity for each effector was determined. Benzoate and cis,cis-muconate competed for the same BenM binding site. However, the conformation changes in BenM caused by benzoate were distinct from those caused by cis,cis-muconate. From these results, a model was proposed to account for BenM's ability to activate benA transcription synergistically in response to both compounds. High-level activation of benA transcription may require a tetramer of BenM to which two monomers bind benzoate and two bind cis,cis-muconate. The structure of this tetramer would be distinct from those bound only to a single type of effector. To better understand these structural differences, a truncated version of BenM was purified and crystallized. X-ray diffraction methods (done in collaboration with Dr. Cory Momany) were used to solve the structure of the BenM effector-binding domain to a resolution of 2.0Å. This study describes the first structural characterization of a protein from an important subclass of LysR-type regulators that controls the catabolism of natural aromatic compounds and pollutants by diverse bacteria.