Amino acid selection during teicoplanin biosynthesis can be switched by a point mutation
Authors: Kittilä Tiia , Cryle Max J. 
Published: November 2023

Glycopeptide antibiotics belong to a diverse class of biologically active compounds that are produced by large enzyme machineries – the non‐ribosomal peptide synthetases (NRPS). Redesigning NRPS systems to produce new chemotherapeutics has fascinating potential but the complexity of the machinery has hampered this potential to date as majority of attempts have yielded a dramatic drop in product yields. A greater understanding of protein interactions, mechanisms and substrate specificities are required if the redesign of NRPS biosynthesis is to be successful. In typical NRPSs multiple active domains are organized into modules, with each module adding one amino acid to the growing peptide chain. Adenylation (A)‐domains select and activate these substrates and as such are the main source of substrate selectivity in the system. Because of this crucial role multiple attempts have been made to change A‐domain selectivity: unfortunately success to date has been limited. So far it is not understood in detail if the reduced product yields are caused by poorly functioning A‐domains or rather by problems in acceptance of the new substrates by following domains such as peptide bond forming condensation‐domains. We are studying biosynthesis of the glycopeptide antibiotic teicoplanin regards to both peptide synthesis and amino acid modification in order to better understand the wide variety of protein‐protein interactions in the NRPS systems. Here, we present our results concerning the substrate specificity of the A‐domain in the third module of teicoplanin NRPS. Selectivity was studied in vitro using a spectroscopic NADH coupled pyrophosphate assay and revealed the hydroxyl groups on the phenylglycine substrate to be crucial for A‐domain activity. A single point mutation in the binding pocket of the A‐domain was enough to change substrate selectivity from the natural 3,5‐dihydroxyphenylglycine to 4‐hydroxyphenylglycine. Further mutations that changed the overall hydrophobicity of the binding pocket strengthened the activation of 4‐hydroxyphenylglycine and remarkably lead to higher enzyme activity than what was seen for the wild type system (kcat/Km (WT) = 65 mM−1 min−1, kcat/Km (mutant) = 390 mM−1 min−1). Methoxy‐phenylglycine substrates were not accepted by any of the mutants tested underlining the necessity of A‐domain interactions with the hydroxyl groups of the amino acid residue. These studies have revealed the basis of phenylglycine selection by A‐domains and more importantly show that substrate selectivity of an A‐domain can be efficiently changed even in a complex NRPS such as the one producing teicoplanin. These results are especially relevant as the amino acid activated by the third module is highly variable between different glycopeptide antibiotic classes and thus modifications on the third module can provide a way to modify the antibiotic activity of teicoplanin.Support or Funding InformationThis work was supported by the Deutsche Akademischer Austausch Dienst (Graduate School Scholarship Program) and the Deutsche Forschungsgemeinschaft (Emmy‐Noether Program).