Abstract
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.
Highlights
Nonribosomal peptides (NRP) represent a very versatile group of low to medium molecular weight compounds that exhibit various biological activities
The Nocardia lactamdurans ACVS was overexpressed in E. coli HM0079 as a C-terminal 6xHis-tagged protein, and purified by Ni2+ affinity purification
We report on the characterization of the Nocardia lactamdurans activity was 0.78 ± 0.14 μM (ACV) synthetase, heterologously overexpressed in E. coli HM0079 [32] and purified to homogeneity
Summary
Nonribosomal peptides (NRP) represent a very versatile group of low to medium molecular weight compounds that exhibit various biological activities. These peptides are exclusively produced by nonribosomal peptide synthetases (NRPS) and do contain proteinogenic amino acids, but may contain a wide variety of non-proteinogenic amino acids and hydroxy acids [1]. NRP synthesis universally starts in every module with the adenylation (A) domain, serving as a highly selective gate keeper, which recruits and adenylates a specific substrate, thereby forming an acyl-adenylate conjugate. The substrate-conjugate is transferred to the thiolation (T) domain by means of the phosphopantetheine (ppant) arm, with the AMP being released. The activated substrates are transported to the donor and acceptor sites of the up- or downstream
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