The role of beta/alpha/beta/beta/beta-module-containing proteins in resistance and metabolism of Pseu-domonas aeruginosa

• Applicant: Professor Dr. Wulf Blankenfeldt
• Subject Area: Structural Biology ; Biochemistry
• Term: from 2013 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 235474366

The genomes of more than 3000 species have been sequenced to date, but a large part of this information cannot be interpreted because the functions of many genes and their prod-ucts are unknown. This annotation problem severely limits our understanding of the mo-lecular 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 will 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 Pseu-domonas aeruginosa. These proteins contain two to four beta/alpha/beta/beta/beta-modules (babbb-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 addi-tion, we identified a group with unknown functions that has not been recognized in the litera-ture. Initial experiments indicate that some babbb-module proteins bind pyocyanin, a toxic phenazine that P. aeruginosa uses as virulence factor, and that at least one of them is in-volved in pyocyanin resistance. Crystal structures of eight of the 22 proteins have been de-termined 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 uncharac-terized P. aeruginosa babbb-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 will use purified recombinant proteins, test them for known activities of other babbb-module proteins and determine their structures for docking calculations to discover potential ligands. Bio-chemical assays and physiological experiments with mutants of P. aeruginosa will be used to corroborate structure-derived hypotheses about functions of the investigated proteins.Because many babbb-module proteins are involved in resistance, the expected insight may also enable new approaches to target infectious diseases.

• DFG Programme: Research Grants