Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Synthetic Biology and Cell Engineering, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
PLoS One. 2020 Apr 10;15(4):e0231290. doi: 10.1371/journal.pone.0231290. eCollection 2020.
The L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS) is a nonribosomal peptide synthetase (NRPS) that fulfills a crucial role in the synthesis of β-lactams. Although some of the enzymological aspects of this enzyme have been elucidated, its large size, at over 400 kDa, has hampered heterologous expression and stable purification attempts. Here we have successfully overexpressed the Nocardia lactamdurans ACVS in E. coli HM0079. The protein was purified to homogeneity and characterized for tripeptide formation with a focus on the substrate specificity of the three modules. The first L-α-aminoadipic acid-activating module is highly specific, whereas the modules for L-cysteine and L-valine are more promiscuous. Engineering of the first module of ACVS confirmed the strict specificity observed towards its substrate, which can be understood in terms of the non-canonical peptide bond position.
L-δ-(α-氨基己二酰基)-L-半胱氨酸-D-缬氨酸合成酶（ACVS）是一种非核糖体肽合成酶（NRPS），在β-内酰胺的合成中起着至关重要的作用。尽管已经阐明了该酶的一些酶学方面，但由于其超过 400 kDa 的巨大尺寸，阻碍了其异源表达和稳定纯化的尝试。在这里，我们成功地在大肠杆菌 HM0079 中过表达了乳酰乳杆菌 ACVS。该蛋白质被纯化至均一性，并对三肽形成进行了特性研究，重点是三个模块的底物特异性。第一个 L-α-氨基己二酸激活模块具有高度特异性，而 L-半胱氨酸和 L-缬氨酸的模块则更具混杂性。对 ACVS 的第一个模块的工程改造证实了对其底物的严格特异性，这可以根据非典型的肽键位置来理解。
