Department of Biochemistry, University of Otago, Dunedin, 9054, New Zealand.
The Ferrier Research Institute, Victoria University of Wellington, Petone, 5046, New Zealand.
Biotechnol Lett. 2021 Jul;43(7):1467-1473. doi: 10.1007/s10529-021-03135-9. Epub 2021 Apr 23.
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.
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.
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.
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。
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。
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。
