Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity

Sci Adv. 2024 Nov 29;10(48):eadp6775. doi: 10.1126/sciadv.adp6775. Epub 2024 Nov 29.

Abstract

Engineering nonribosomal peptide synthetases (NRPSs) has been a "holy grail" in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the "gate-keeper" adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

MeSH terms

  • Kinetics
  • Models, Molecular
  • Mutation
  • Oxidoreductases* / chemistry
  • Oxidoreductases* / genetics
  • Oxidoreductases* / metabolism
  • Peptide Synthases* / chemistry
  • Peptide Synthases* / genetics
  • Peptide Synthases* / metabolism
  • Protein Engineering
  • Substrate Specificity

Substances

  • Peptide Synthases
  • carboxylic acid reductase
  • Oxidoreductases
  • non-ribosomal peptide synthase