Biochemical characterization of the Nocardia lactamdurans ACV synthetase

• Published in: PloS one Vol. 15; no. 4; p. e0231290
• Main Authors: Iacovelli, Riccardo, Zwahlen, Reto D, Bovenberg, Roel A L, Driessen, Arnold J M
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.04.2020; Public Library of Science (PLoS)
• Subjects: Amino acids; Biology and Life Sciences; Biotechnology; Chromatography; E coli; Engineering and Technology; Enzymes; Homogeneity; Medicine and Health Sciences; Modules; Nocardia; Peptides; Proteins; Research and Analysis Methods; Substrate specificity; Substrates; Valine; β-Lactam antibiotics; Netherlands
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0231290

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.