The role of βαβββ-module-containing proteins in resistance and metabolism of Pseudomonas aeruginosa
The genomes of several thousand species have been sequenced to date, but a large part of this information cannot be interpreted because the functions of many genes and their products are unknown. This “annotation problem” severely limits our understanding of the molecular basis of life and restricts the use of the available data in biotechnology and medicine.
A reliable annotation solely from computational sequence analysis is impossible at present, and different experimental techniques have to be used synergistically for the characterization of gene function. Here, we combine in silico analysis with biochemical, structural, and microbiological experiments to elucidate the functions of all members of the “glyoxalase-I/bleomycin resistance protein” family found in the genome of the bacterial pathogen Pseudomonas aeruginosa. These proteins contain two to four βαβββ-modules, and only four of the 22 genes encoding these proteins in P. aeruginosa are characterized. Sequence analysis revealed that the targeted proteins encompass metal-dependent “vicinal oxygen chelate” enzymes and several proteins that sequester aromatic compounds. In addition, we identified a group with unknown functions that has not been recognized in the literature. Our initial experiments indicated that some βαβββ-module proteins bind pyocyanin, a toxic phenazine that P. aeruginosa uses as virulence factor, and that at least one of them is involved in pyocyanin resistance. Crystal structures of most of the 22 proteins have been determined by us or others, and crystals of two more have been obtained in our group.
Our objectives in this project are (i) to elucidate the molecular functions of the 18 uncharacterized P. aeruginosa βαβββ-module proteins and to understand the underlying activity and selectivity mechanisms, (ii) to determine their physiological roles, and (iii) to use these results to annotate related genes from other microbial species. To arrive at these goals, we use purified recombinant proteins, test them for known activities of other βαβββ-module proteins and determine their structures for docking calculations to discover potential ligands. Biochemical assays and physiological experiments with mutants of P. aeruginosa are used to corroborate structure-derived hypotheses about functions of the investigated proteins.Because many βαβββ-module proteins are involved in resistance, the expected insight may also enable new approaches to target infectious diseases.
- Structure and Function of Proteins - Prof. Wulf Blankenfeldt
DFG - German Research Foundation