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

• Published in: MicrobiologyOpen (Weinheim) Vol. 10; no. 1; pp. e1145 - n/a
• Main Authors: Iacovelli, Riccardo, Mózsik, László, Bovenberg, Roel A.L., Driessen, Arnold J.M.
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01.01.2021; John Wiley and Sons Inc; Wiley
• Subjects: 2-Aminoadipic Acid - metabolism; Amino Acid Sequence; Amycolatopsis - enzymology; Amycolatopsis - genetics; Amycolatopsis - metabolism; Anti-Bacterial Agents - biosynthesis; Anti-Bacterial Agents - metabolism; Antibiotics; beta-Lactams - metabolism; Biosynthesis; Biosynthetic Pathways - genetics; Biosynthetic Pathways - physiology; Carboxyl group; Catalysis; Cloning; Cysteine - chemistry; Domains; Enzymes; Fungi; Genetic Variation - genetics; In vivo methods and tests; Modules; Nocardia lactamdurans; nonribosomal peptide synthetases; Original; Penicillin; penicillin biosynthesis; Penicillium - enzymology; Penicillium - genetics; Penicillium - metabolism; Penicillium rubens; Peptide Synthases - genetics; Peptides; Pharmaceuticals; Plasmids; Protein Domains - genetics; Proteins; Substrate specificity; Substrates; Valine; Nocardia lactamdurans; Penicillium rubens; penicillin biosynthesis; antibiotics; nonribosomal peptide synthetases
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.1145

The l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine synthetase (ACVS) is a trimodular nonribosomal peptide synthetase (NRPS) that provides the peptide precursor for the synthesis of β‐lactams. The enzyme has been extensively characterized in terms of tripeptide formation and substrate specificity. The first module is highly specific and is the only NRPS unit known to recruit and activate the substrate l‐α‐aminoadipic acid, which is coupled to the α‐amino group of l‐cysteine through an unusual peptide bond, involving its δ‐carboxyl group. Here we carried out an in‐depth investigation on the architecture of the first module of the ACVS enzymes from the fungus Penicillium rubens and the bacterium Nocardia lactamdurans. Bioinformatic analyses revealed the presence of a previously unidentified domain at the N‐terminus which is structurally related to condensation domains, but smaller in size. Deletion variants of both enzymes were generated to investigate the potential impact on penicillin biosynthesis in vivo and in vitro. The data indicate that the N‐terminal domain is important for catalysis. A conserved domain was identified in the first module of the l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine synthetase, the first enzyme of the biosynthetic pathway of penicillin. The domain is structurally related to condensation domains, but it is approximately half the size and it lacks the typical active site motif. Nevertheless, its presence is important for catalysis.