STRUCTURAL STUDIES OF NON-RIBOSOMAL PEPTIDE SYNTHETASES, AMBB AND AMBE, IN PSEUDOMONAS AERUGINOSA

Abstract

Non-ribosomal peptide synthetases (NPRS), commonly found in bacteria and fungi, are mega enzymes catalysing the biosynthesis of natural peptides, many of which have medicinal importance. In this work, we undertook structural studies of the NRPS AmbB and AmbE, in P. aeruginosa, which are involved in the biosynthesis of the antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB). The two structures, determined at 2.2 Å and 2.5 Å resolution, include Phosphopantetheine (Ppant) ligand, and a loaded Ppant ligand with L-Alanine, respectively. Both structures illustrate the T domain in a catalytically relevant conformation, with the Ppant arm, interacting with the C domain at the donor site. Several single-site mutations were generated, and the in vivo production of AMB was assessed, in order to probe the interaction of Ppant-L-Alanine in the C domain of AmbB. The condensation activity of the AmbB variants were further confirmed, in vitro, using a radioactivity experiment.