单点突变将 7-氨基头孢烷酸酰化酶转化为 N-酰基高丝氨酸内酯酶。
A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase.
机构信息
Department of Biochemistry, University of Otago, Dunedin, 9054, New Zealand.
The Ferrier Research Institute, Victoria University of Wellington, Petone, 5046, New Zealand.
出版信息
Biotechnol Lett. 2021 Jul;43(7):1467-1473. doi: 10.1007/s10529-021-03135-9. Epub 2021 Apr 23.
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.
目的
通过定点突变改变戊二酰-7-氨基头孢烷酸酰化酶（GCA）对 N-酰基高丝氨酸内酯（AHLs；群体感应信号分子）的特异性。
结果
通过对现有晶体结构的分析，确定了 7 个残基可能是决定底物特异性的关键因素。对这七个选定位置中的每一个都创建了饱和突变文库。对每个文库的高通量活性筛选鉴定出两种变体-Arg255Ala，Arg255Gly-对 N-酰基高丝氨酸内酯底物具有新的活性。Arg255Gly 突变的结构建模表明，与野生型酶中的精氨酸相比，甘氨酸的侧链较小（与 N-酰基高丝氨酸内酯底物的酰基基团）避免了关键的冲突。
结论
突变单个氨基酸残基成功地将 GCA（对 AHLs 没有检测到活性）转化为 AHL 酰化酶。这种方法可能对进一步工程化“群体感应淬灭”酶有用。
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