Biochemical characterization of the Nocardia lactamdurans ACV synthetase

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 acidactivating 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.

Identification of a conserved N‐terminal domain in the first module of ACV synthetases

“…The product was checked by restriction analysis and sequencing (Macrogen Europe B.V.). Lastly, the pBAD‐ Nl ΔC*‐ACVS plasmid was assembled using the Golden Gate assembly reaction (Engler et al, 2009) as described previously (Iacovelli et al, 2020) and cloned into DH10β chemically competent E. coli cells for storage.…”

“…The main residues of core motifs C1-C7 of the condensation domain family are highlighted in red. The active site motif, C3, and motifs C1-C5 are completely lacking in both C* domains and ADA, while C6 is retained only in the case of NPS3 and C7 in all cases been extensively characterized as a purified protein in previous studies (Coque et al, 1996;Iacovelli et al, 2020). To generate the Nl ACVS ΔC* variant, we employed a Golden Gate-based assembly strategy previously described (Iacovelli et al, 2020).…”

“…The active site motif, C3, and motifs C1-C5 are completely lacking in both C* domains and ADA, while C6 is retained only in the case of NPS3 and C7 in all cases been extensively characterized as a purified protein in previous studies (Coque et al, 1996;Iacovelli et al, 2020). To generate the Nl ACVS ΔC* variant, we employed a Golden Gate-based assembly strategy previously described (Iacovelli et al, 2020). The construct was subsequently cloned and overexpressed in E. coli HM0079 (Gruenewald et al, 2004), a strain that natively expresses a phosphopantetheinyl transferase, responsible for the conversion of apo-PCP to holo-PCP domains, necessary for the function of NRPS.…”

“…Because of the relevance of the β-lactam biosynthetic pathway and the extensive research covering it, the ACVS became soon a model to investigate nonribosomal peptide biosynthesis. The protein has been studied in filamentous fungi such as Penicillium, Aspergillus, and Cephalosporium as well as bacterial Nocardia and Streptomyces species, both in vivo and in vitro (Baldwin et al, 1990(Baldwin et al, , 1991Coque et al, 1996;Iacovelli et al, 2020;Jensen et al, 1990;van Liempt et al, 1989;Schwecke et al, 1992;Tahlan et al, 2017;Theilgaard et al, 1997). Despite the lack of 3D structures, it is well known that the ACVS is a trimodular NRPS with a reported canonical domain arrangement: AT 1 -CAT 2 -CATETe 3 .…”

“…Interestingly, the adenylation domain of the first module of ACVS is the only NRPS domain that is known to activate and utilize l-α-aminoadipic acid as substrate (Flissi et al, 2020). Besides, l-α-Aaa is adenylated on the δ-carboxyl group, resulting in a non-canonical peptide bond formation between l-α-Aaa and l-Cys (Iacovelli et al, 2020), suggesting a substrate recognition mechanism different from that of canonical amino acid-activating adenylation domains (Conti et al, 1996(Conti et al, , 1997Stachelhaus et al, 1999).…”