Biochemical characterization of the Nocardia lactamdurans ACV synthetase

Riccardo Iacovelli; Reto D. Zwahlen; Roel A.L. Bovenberg; Arnold J.M. Driessen

doi:10.1371/journal.pone.0231290

Biochemical characterization of the Nocardia lactamdurans ACV synthetase

Riccardo Iacovelli
, Reto D. Zwahlen
, Roel A.L. Bovenberg
, Arnold J.M. Driessen^*

^*Corresponding author for this work

Not published at VTT

University of Groningen

Research output: Contribution to journal › Article › Scientific › peer-review

5 Citations (Scopus)

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.

Original language	English
Article number	e0231290
Journal	PLoS ONE
Volume	15
Issue number	4
DOIs	https://doi.org/10.1371/journal.pone.0231290
Publication status	Published - Apr 2020
MoE publication type	A1 Journal article-refereed

Funding

We declare an affiliation with DSM Biotechnology (Delft, The Netherlands) for the period of the study. DSM Biotechnology provided support in the form of salary for author R. A. L. Bovenberg, who is both associated with the University of Groningen and the company, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the author are articulated in the 'author contributions' section. We thank DSM Sinochem Pharmaceuticals (now Centrient Pharmaceuticals) for the Nl ACVS construct, and in particular Jan-Metske van der Laan for the scientific support. We also would like to thank Susan Fekken for her help with the kinetics experiments.

Keywords

Amino Acid Sequence
Nocardia/enzymology
Peptide Synthases/chemistry
Peptides/chemistry
Protein Domains
Protein Engineering
Substrate Specificity

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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.",

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N2 - 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.

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Biochemical characterization of the Nocardia lactamdurans ACV synthetase

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