In-vitro Characterization of an L-Kynurenine-Responsive Transcription Regulator of the Oxidative Tryptophan Degradation Pathway in Burkholderia xenovorans

Abstract

To study the transcriptional regulation of oxidative tryptophan degradation in Burkholderia, we used comparative genomics that focused on the operon containing the genes annotated as kynA, kynU and kynB. In all sequenced Beta-proteobacteria to-date, including Burkholderia, Ralstonia, Collimonas, and Cupriavidus species, there is a conserved AsnC/Lrp family transcriptional regulator (TR) gene located upstream and in the opposite strand of the operon encoding for the oxidative tryptophan degradation genes. In Burkholderia xenovorans the TR is Bxe_A0736. GST-Bxe_A0736 binds L-kynurenine with greater affinity and specificity than any other amino acid or tryptophan degradation product with a dissociation constant of ~82 ± 11 ?M. DNaseI footprinting suggested that Bxe_A0736 protects a set of four degenerate, palindromic sequences within the intergenic region between Bxe_A0735 (kynB) and Bxe_A0736. The optimal consensus sequence obtained by analysis for these sites, ATATTCCGAATAT, closely resembles the sequence obtained with a protein binding microarray. Under our fluorescence anisotropy experimental conditions, 1 mM L-kynurenine increased the affinity of Bxe_A0736 for a portion of its promoter region. Our results are consistent with Bxe_A0736 acting as the TR that promotes the transcription of the oxidative tryptophan degradation genes in the presence of L-kynurenine while inhibiting the transcription of its own gene.