The second paper from Lygie Esquirol's PhD is now available as a paper-in-press at the Journal of Biological Chemistry.
The work described in this paper is the latest in a series on pieces in which we investigate the enzymes involved in degrading the herbicide atrazine. Like many herbicides and other pesticides, the use of atrazine contributes to high yields in broad acre cropping; however, atrazine has a somewhat mixed reputation, and so it's important to know about its fate in the environment.
The common soil bacterium Pseudomonas has evolved a catabolic pathway that lets it 'eat' atrazine - using it as a nitrogen source. This work contributes to our understanding of how Pseudomonas has evolved this pathway, and more broadly how bacteria evolve new metabolic functions in general.
In this case, we were able to deduce the likely ancestor of AtzE (1-carboxybiuret amidohydrolase) from the presence of a small, unexpected ancillary protein (AtzG - the titular appendix!) that appears to be a the remaining vestige of a larger complex (the transamidosome - essential for making certain charged tRNAs). The picture shows how well the two complexes align - even internal structures (like tunnels) are conserved.
We discovered AtzG by determining the structure of AtzE purified from Pseudomonas (thanks to Janet and Tom at C3, @CSIROC3) and deriving the amino acid sequence of AtzG from that structure.
Finding AtzG also alerted us to the presence of another small, previously unidentified protein in the atrazine catabolism pathway, AtzH, which we're currently investigating.
AtzG and AtzH are quite small for proteins (68 and 120 amino acids, respectively) and one of the reasons that they were overlooked is that their genes are so short that they were ignored during annotation - so one take-home message here is that even tiny open-reading frames can be important.