Teicoplanin Nonribosomal Peptide Synthetase Is Unable to Incorporate Alpha-Ketoacid Building Blocks

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Synthesis

Background:

• Glycopeptide antibiotics (GPAs) are crucial last-resort treatments against multidrug-resistant bacteria.
• Nonribosomal peptide synthetases (NRPSs) assemble these complex molecules, with adenylation (A) domains controlling substrate selection and activation.
• The initial building block in GPAs can be an amino acid or ketoacid, posing questions about A-domain specificity.

Purpose of the Study:

• To investigate the substrate acceptance of the teicoplanin NRPS initiation module, specifically its A-domain.
• To determine if this A-domain can incorporate non-amino acid substrates like ketoacids.
• To elucidate the structural basis for substrate selectivity in GPA biosynthesis.

Main Methods:

• In vitro biochemical assays to test substrate activation and loading.
• Structural characterization of the teicoplanin A-domain in complex with a substrate.
• Molecular dynamics simulations to analyze substrate interactions and catalytic mechanisms.

Main Results:

• The teicoplanin NRPS A-domain showed a preference for activating ketoacids over its native amino acid, l-Hpg.
• Despite ketoacid activation, only amino acids (d/l)-Hpg and related compounds were loaded onto the carrier protein.
• Structural analysis revealed altered substrate carboxylate positioning, explaining high pyrophosphate release with d-Hpg.

Conclusions:

• Ketoacid incorporation in GPA biosynthesis likely occurs post-amino acid incorporation by the NRPS.
• A-domain specificity is determined by both substrate activation and carrier protein loading steps.
• Understanding these dual roles is critical for deciphering NRPS substrate selectivity and engineering novel GPAs.