Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis

Abstract

Nonribosomal peptide synthetases (NRPSs) are a family of multidomain, multimodule enzymes that synthesize structurally and functionally diverse peptides, many of which are of great therapeutic or commercial value. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In many NRPS modules, the C domain is replaced by the heterocyclization (Cy) domain, a homologous domain that performs two consecutive reactions by using hitherto unknown catalytic mechanisms. It first catalyzes amide bond formation, and then the intramolecular cyclodehydration between a Cys, Ser, or Thr side chain and the backbone carbonyl carbon to form a thiazoline, oxazoline, or methyloxazoline ring. The rings are important for the form and function of the peptide product. We present the crystal structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to probe the roles of active-site residues by using mutational analyses in a peptide synthesis assay with intact bacillamide synthetase. The drastically different effects of these mutants, interpreted by using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain and implicate a previously unexamined Asp-Thr dyad in catalysis of the cyclodehydration reaction.nonribosomal peptide synthetase | heterocyclization domain | X-ray crystallography | bacillamide | mutational analysis

The Biosynthesis of the Benzoxazole in Nataxazole Proceeds via an Unstable Ester and has Synthetic Utility

“…The spontaneous conversion of 1 is predicted to occur via a tetrahedral hemiorthoamide intermediate formed by attack of the amine on the ester (Figure a). In RiPP and NRP systems, oxazoline biosynthesis also proceeds via this hemiorthoamide intermediate (phosphorylated in RiPP) but it is formed by the attack of hydroxyl upon an amide (Figure b).…”

“…Unconjugated oxazoles are mostly produced by ribosomally synthesized post‐translationally modified peptides (RiPPs) synthesis and by non‐ribosomal peptides (NRPs) synthesis . Oxazoles in both families derive from nucleophilic attack of the hydroxyl group of Ser(Thr) upon amide bonds through a common hemiorthoamide intermediate (Figure b), but via distinct enzymatic routes . Two polyketide synthase–NRPS hybrid natural products, oxazolomycin and conglobatin, have been proposed to utilize an N‐type ATP pyrophosphohydrolase to form oxazoles .…”

The Biosynthesis of the Benzoxazole in Nataxazole Proceeds via an Unstable Ester and has Synthetic Utility

“…[38] Thespontaneous conversion of 1 is predicted to occur via at etrahedral hemiorthoamide intermediate formed by attack of the amine on the ester (Figure 6a). In RiPP [24] and NRP [29][30][31][32] systems,o xazoline biosyn-thesis also proceeds via this hemiorthoamide intermediate (phosphorylated in RiPP) but it is formed by the attack of hydroxyl upon an amide (Figure 1b).…”

“…Unconjugated oxazoles are mostly produced by ribosomally synthesized post‐translationally modified peptides (RiPPs) synthesis and by non‐ribosomal peptides (NRPs) synthesis . Oxazoles in both families derive from nucleophilic attack of the hydroxyl group of Ser(Thr) upon amide bonds through a common hemiorthoamide intermediate (Figure b), but via distinct enzymatic routes . Two polyketide synthase–NRPS hybrid natural products, oxazolomycin and conglobatin, have been proposed to utilize an N‐type ATP pyrophosphohydrolase to form oxazoles .…”

“…[20,21] Oxazoles in both families derive from nucleophilic attack of the hydroxyl group of Ser(Thr) upon amide bonds [20,22] through ac ommon hemiorthoamide intermediate ( Figure 1b), but via distinct enzymatic routes. [23][24][25][26][27][28][29][30][31][32][33][34] Tw o polyketide synthase-NRPS hybrid natural products,o xazolomycin and conglobatin, have been proposed to utilize an Ntype ATPpyrophosphohydrolase to form oxazoles. [35,36] None of these enzymes have been reported to catalyze the formation of benzoxazole and such activity would seem unlikely based on their known substrates.…”