Inherited retinal diseases (IRDs) are rare disorders, typically presenting as Mendelian traits, that result in stationary or progressive visual impairment. They are characterized by extensive genetic ...

Non-ribosomal peptide synthetases (NRPSs) are megaenzymes responsible for the biosynthesis of many clinically important natural products, from early modern medicines (penicillin, bacitracin) to current blockbuster drugs (cubicin, vancomycin) and newly approved therapeutics (rezafungin)1,2. The key chemical step in these biosyntheses is amide bond formation between aminoacyl building blocks, catalysed by the condensation (C) domain3. There has been much debate over the mechanism of this reaction3–12. NRPS condensation has been difficult to fully characterize because it is one of many successive reactions in the NRPS synthetic cycle and because the canonical substrates are each attached transiently as thioesters to mobile carrier domains, which are often both contained in the same very flexible protein as the C domain. Here we have produced a dimodular NRPS protein in two parts, modified each with appropriate non-hydrolysable substrate analogues13,14, assembled the two parts with protein