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，但其中很大一部分信息无法解释，因为许多基因及其产物的功能尚不清楚。这一问题严重限制了我们对生命的分子基础的理解，并限制了现有数据在生物技术和医学中的应用，目前仅通过计算序列分析无法进行可靠的注释，必须综合使用不同的实验技术来表征基因功能。在这里，我们将联合收割机在硅片分析与生物化学，结构和微生物实验，以阐明所有成员的glycosidase-I/博来霉素耐药蛋白家族中发现的细菌病原体铜绿假单胞菌的基因组中的功能。这些蛋白质含有2至4个β/α/β-模块（babbb-模块），并且在铜绿假单胞菌中编码这些蛋白质的22个基因中只有4个被表征。序列分析表明，目标蛋白包括金属依赖性邻位氧螯合酶和几种螯合芳香族化合物的蛋白质。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模块蛋白结合绿脓菌素，绿脓菌素是铜绿假单胞菌用作毒力因子的有毒吩嗪，并且其中至少一种涉及绿脓菌素抗性。其中8种蛋白质的晶体结构已被我们或他人测定，另外2种蛋白质的晶体结构已被我们组获得。我们在这个项目中的目标是（i）阐明18个未表征的铜绿假单胞菌babbb模块蛋白的分子功能，并了解潜在的活性和选择性机制，（ii）确定它们的生理作用，以及（iii）使用这些结果注释来自其他微生物物种的相关基因。为了达到这些目标，我们将使用纯化的重组蛋白，测试它们的其他巴布模块蛋白的已知活性，并确定它们的结构，用于对接计算，以发现潜在的配体。铜绿假单胞菌突变体的生化分析和生理实验将用于证实有关所研究蛋白质功能的结构衍生假说。由于许多babbb模块蛋白参与抗性，预期的洞察力也可能使新的方法靶向感染性疾病。

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.