Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module

Abstract

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

Bacillamide F, Extracted from Marine Bacillus atrophaeus C89, Preliminary Effects on Leukemia Cell Lines

“…We have predicted that Oxidase encoded by oxidase gene located upstream of nrps gene cluster could catalyze AlaCys thiazoline to AlaCys thiazole [ 19 ]. The dehydrogenation activity of Oxidase has been further confirmed in the peptide intermediate while it is covalently attached to the NRPS [ 29 ]. In this study, addition of L-proline into a culture medium of B. atrophaeus C89 increased the production of bacillamide F ( Figure 2 ).…”

Understanding the Diversity and Molecular Basis of Biosynthesis of Heterocyles in Natural Products Produced by Actinobacteria

“…there are two more examples of NRPSs with dimeric structures whose structures were solved by cryo-EM analysis to date. 55,56 Based on this structural information of FmoA3, the structure of the active site was compared with the structure of the Cy domain, which has already been reported to catalyze both peptide bond formation and heterocyclization reactions. 38,39 However no special features were found near the active site.…”

“…Iminimycin (55) is a compound isolated from Streptomyces griseus OS-3601 by a group at Kitasato University, and two derivatives are known (iminimycin A [55] and B [60], Figure 19). 68,69 Both have an iminium cation and a 6-5-3 tricyclic structure.…”

Nonribosomal Peptide Synthesis

“…Non-ribosomal peptide synthetases (NRPSs) enzymes are poly-functional mega-synthases that biosynthesize NRPs [7,9,10]. NRPSs, multi-modular enzyme or enzyme complexes from common bacteria, less common eukarya, and rare archaea, are capable of producing a wide range of natural pharmaceutical products (Bacitracin, antibiotics for skin infections; Bleomycin antitumor; Cyclosporin, antifungal, and immunosuppressive drugs; Daptomycin, antibiotics) [5,7,11].…”