Many bacteria use membrane-diffusible small molecule quorum signals to coordinate gene transcription in response to changes in cell density, known as quorum sensing (QS). Among these, acyl-homoserine lactones (AHL) are widely distributed in Proteobacteria and are involved in controlling the expression of virulence genes and biofilm formation in pathogens, such as Pseudomonas aeruginosa. AHL molecules are specifically biosynthesized by the cognate LuxI type AHL synthases using S-adenosylmethionine (SAM) and either acyl carrier protein (ACP)- or CoA-coupled fatty acids through a two-step reaction. Here, we characterize a CoA-dependent LuxI synthase from Rhodopseudomonas palustris that utilizes an aryl-CoA substrate that is environmentally derived, specifically p-coumaric acid. We leverage structures of this aryl-CoA-dependent synthase, along with our prior studies of an acyl-CoA-dependent synthase, to identify residues that confer substrate chain specificity in these enzymes. We test our predictions by carrying out biochemical, kinetic, and structural characterization of representative AHL signal synthases. Our studies provide an understanding of various AHL synthases that may be deployed in synthetic biological applications and inform on the design of specific small molecule therapeutics that can restrict virulence by targeting quorum signaling.

中文翻译：

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法定信号合酶特异性的结构指导生化分析。

许多细菌使用膜可扩散的小分子法定信号来协调基因转录，以响应细胞密度的变化，称为法定感应（QS）。其中，酰基高丝氨酸内酯（AHL）广泛分布在变形杆菌中，并参与控制病原体如铜绿假单胞菌中毒力基因的表达和生物膜的形成。通过S-腺苷甲硫氨酸（SAM）和酰基载体蛋白（ACP）或CoA偶联的脂肪酸通过两步反应，通过同源LuxI型AHL合成酶特异性地生物合成AHL分子。在这里，我们表征了来自红假单胞菌的CoA依赖性LuxI合酶。该方法利用了环境衍生的芳基-CoA底物，特别是对-香豆酸。我们利用这种芳基-CoA依赖合酶的结构，以及我们先前对酰基-CoA依赖合酶的研究，来确定赋予这些酶底物链特异性的残基。我们通过进行代表性AHL信号合酶的生化，动力学和结构表征来检验我们的预测。我们的研究提供了对可能在合成生物学应用中部署的各种AHL合成酶的了解，并为特定小分子疗法的设计提供了信息，这些疗法可以通过靶向群体信号来限制毒力。