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Abstract：
Bacterial type II phosphopantetheinyl transferases (PPTases), required for the activation of many cellular mega-synthases, have been validated as promising drug targets in several pathogens. Activation of the blue-pigment-synthesizing nonribosomal peptide synthetase BpsA by a target PPTase can be used to screen in vitro for new antibiotic candidates from chemical libraries. For a complete screening platform, there is a need to also counter-screen inhibitors for cross-reactivity with the endogenous human Type II PPTase (hPPTase), as this is a likely source of toxicity. As hPPTase is unable to recognize the PCP-domain of native BpsA, we used a combination of directed evolution and rational engineering to generate a triple-substitution variant that is able to be efficiently activated by hPPTase. Our engineered BpsA variant was able to readily detect inhibition of both hPPTase and the equivalent rat PPTase by broad-spectrum PPTase inhibitors, demonstrating its potential for high-throughput counter-screening of novel antibiotic candidates.
摘要：
细菌II型磷酸蒽环酰转移酶（PPTases），需要激活许多细胞巨合酶，已被验证为几种病原体的有希望的药物靶标。目标PPTase激活合成蓝色色素的非核糖体肽合成酶BpsA可用于从化学文库中体外筛选新的抗生素候选物。对于一个完整的筛选平台，还需要反筛选与内源性人II型PPTase（hPPTase）交叉反应性的抑制剂，因为这可能是毒性的来源。由于hPPTase无法识别天然BpsA的PCP结构域，我们使用定向进化和合理工程的组合来产生能够被hPPTase有效激活的三重置换变体。我们的工程BpsA变体能够容易地检测到广谱PPTase抑制剂对hPPTase和等效大鼠PPTase的抑制作用，证明了其对新型候选抗生素的高通量反筛选的潜力。
