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

迄今为止，已经对3000多个物种的基因组进行了测序，但由于许多基因及其产物的功能尚不清楚，因此大部分信息无法解释。这种注释问题严重限制了我们对生命分子基础的理解，并限制了生物技术和医学中现有数据的使用。目前，仅从计算序列分析中获得可靠的注释是不可能的，并且必须协同使用不同的实验技术来表征基因功能。在这里，我们将结合计算机分析、生化、结构和微生物学实验来阐明细菌病原体铜绿假单胞菌基因组中发现的乙草酸酶- 1 /博来霉素耐药蛋白家族所有成员的功能。这些蛋白含有2到4个β / α / β / β / β模块（babbb-模块），在铜绿假单胞菌中编码这些蛋白的22个基因中只有4个被鉴定出来。序列分析表明，目标蛋白包含金属依赖的邻氧螯合酶和几种分离芳香族化合物的蛋白。此外，我们还发现了一组在文献中未被识别的未知功能。最初的实验表明，一些babbb-模块蛋白结合pyocyanin（一种有毒的非那嗪，被铜绿假单胞菌用作毒力因子），并且其中至少有一种与pyocyanin耐药性有关。22种蛋白质中有8种的晶体结构已经由我们或其他人确定，我们小组还获得了另外两种的晶体。本项目的目标是：(1)阐明18个未鉴定的P. aeruginosa babbb-模块蛋白的分子功能，了解其潜在的活性和选择性机制；(2)确定其生理作用；(3)利用这些结果对其他微生物物种的相关基因进行注释。为了达到这些目标，我们将使用纯化的重组蛋白，测试它们对其他babbb模块蛋白的已知活性，并确定它们的结构进行对接计算，以发现潜在的配体。对铜绿假单胞菌突变体的生化分析和生理实验将用于证实结构衍生的关于所研究蛋白质功能的假设。由于许多babbb-模块蛋白参与了耐药性，预期的发现也可能使针对传染病的新方法成为可能。

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.