Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase

While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria. Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria. Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway. This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans. Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R. metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU). Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.