Discovery and characterization of an L‐kynurenine responsive transcriptional regulator of the oxidative tryptophan degradation pathway in Burkholderia xenovorans

Abstract

To uncover the transcriptional regulation of oxidative tryptophan degradation, comparative genomics were used which focused on the operon containing the genes kynA, kynU and kynB. In β and γ‐proteobacteria, including Burkholderia, Ralstonia, Collimonas, Cupriavidus and Pseudomonas species, there is a conserved asnC/lrp family transcriptional regulator (TR) gene located upstream of the operon on the opposite strand. In Burkholderia xenovorans the TR is Bxe_A0736 and we designate it as kynR. Using DNAse footprinting with fluorescence capillary electrophoresis, we determined that the KynR protein binds a set of four degenerate, palindromic sequences within the intergenic region from 35 bp upstream of kynB to over the start site of kynR. The optimal consensus sequence is ATATTCCGAATAT. KynR binds L‐kynurenine with greater affinity than any other amino acid or tryptophan degradation product and does so with a dissociation constant of 67 ± 14 μM, as determined by frontal affinity chromatography with ESI‐MS detection (FAC‐MS). Using fluorescence anisotropy, we determined that L‐kynurenine increases the affinity of KynR for a 39 bp section of the regulatory site from 145 +/− 2 nM to 97 +/− 1 nM. We propose that KynR promotes the transcription of kynA, kynU and kynB in the presence of L‐kynurenine while inhibiting the transcription of its own gene, kynR. This research was funded by the DOE/LANL LDRD program.