Isolation and characterization of mutations in the Escherichia coli regulatory protein XapR

Abstract

In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase in Escherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

FIG. 1

Inducer-binding regions in XapR as defined by the isolated point mutations (shown above the figure). The regions proposed by Schell (21) are shown as grey boxes. Also shown is the proposed DNA-binding region at the N-terminal part of the protein.

FIG. 2

Structure of the C-terminal fragment of CysB from K. aerogenes (25) modelled with XapR mutations. The positions in CysB (amino acids 99, 128, 199, 201, 203, and 206 [highlighted]) that correspond to the mutations in XapR were found by ClustalW sequence alignment of the two amino acid sequences (accession no. P45600 [CysB] and P23841 [XapR]). The sulfate ions believed to be positioned in the inducer-binding domain are shown. (A) Dimeric form of CysB(88–324). (B) The molecule is turned 90° relative to that in panel A, and only the left subunit is shown.