Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis.
Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis.
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DOI：
10.1021/bi500026e
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发表时间：
2014-05-20
期刊：
影响因子：
2.9
通讯作者：
Kohli RM
中科院分区：
文献类型：
--
作者：
Mo CY;Birdwell LD;Kohli RM
细菌利用严格调控的应激反应(SOS)途径对包括抗菌药在内的各种遗传毒性物质做出反应。SOS反应的激活受一种关键的抑制物--蛋白水解酶LexA的调节，LexA在应激状态下经历自身的蛋白分解，导致SOS基因的去抑制。值得注意的是，在感染模型中，LexA自裂解活性的遗传失活显著降低了获得性抗生素耐药性，并使细菌对传统抗生素高度敏感，这表明对LexA的机制研究可能有助于了解其作为对抗获得性耐药性的新靶点的生存能力。尽管对LexA的结构有深入的了解，但对该酶的蛋白酶专一性缺乏详细的了解。在这里，我们使用饱和和位置扫描突变LexA的内部裂解区域来分析>；140突变，并从人类病原体铜绿假单胞菌(LexAPa)产生全面的LexA特异性图谱。我们发现LexAPA活性中心具有独特的底物识别模式。位置P1-P3倾向于与活性部位有特定接触的小疏水残基，而位置P5和P1‘倾向于柔性甘氨酸残基，这可能有助于构象变化，从而允许自蛋白降解。我们进一步表明，在切割区域内稳定β-TURN可以增强LexA的自我蛋白分解活性。最后，我们确定了剪切键(P4和P2‘)两侧的允许位置，这些位置对广泛的突变是耐受的。我们的研究揭示了LexA自体蛋白水解酶的活性部位结构，并为设计SOS途径的探针提供了见解。
Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA’s self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme’s protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA’s internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1–P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1′ show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2′) that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.
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影响因子：
64.8
作者：
Beaber, JW;Hochhut, B;Waldor, MK
通讯作者：
Waldor, MK
DOI：
10.1126/science.1176667
发表时间：
2009-08-28
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Fischbach MA;Walsh CT
通讯作者：
Walsh CT
影响因子：
64.5
作者：
Boshoff, HIM;Reed, MB;Mizrahi, V
通讯作者：
Mizrahi, V
影响因子：
56.9
作者：
BOWIE, JU;REIDHAAROLSON, JF;SAUER, RT
通讯作者：
SAUER, RT
影响因子：
2.7
作者：
Gotoh, H;Kasaraneni, N;Weitao, T
通讯作者：
Weitao, T