Objective

To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis.

Results

Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate.

Conclusions

Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

中文翻译：

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单点突变将戊二酰-7-氨基头孢烷酸酰化酶转化为 N-酰基-高丝氨酸内酯酰化酶

客观的

通过定点诱变改变戊二酰-7-氨基头孢烷酸酰化酶 (GCA) 对N-酰基高丝氨酸内酯（AHL；群体感应信号分子）的特异性。

结果

通过分析现有的晶体结构作为底物特异性的潜在决定因素鉴定了七个残基。为七个选定位置中的每一个创建位点饱和诱变文库。每个文库的高通量活性筛选确定了两个变体——Arg255Ala、Arg255Gly——对N-酰基高丝氨酸内酯底物具有新的活性。Arg255Gly 突变的结构模型表明，甘氨酸的较小侧链（与野生型酶中的精氨酸相比）避免了与N-酰基高丝氨酸内酯底物的酰基发生关键冲突。

结论

单个氨基酸残基的突变成功地将 GCA（对 AHL 没有可检测的活性）转化为 AHL 酰基转移酶。这种方法可用于“群体淬灭”酶的进一步工程化。