Oxidation of the Cyanobactin Precursor Peptide Is Independent of the Leader Peptide and Operates in a Defined Order

Abstract

The five-membered nitrogen plus heteroatom rings known as azolines or in their oxidized form as azoles are very common in natural products and drugs. The oxidation of thiazoline to thiazole in the cyanobactin class of natural products is one of the several important transformations that are known to alter the biological properties of the compound. The ordering of the various chemical reactions that occur during cyanobactin biosynthesis is not fully understood. The structure of the flavin-dependent enzyme responsible for the oxidation of multiple thiazolines reveals it contains an additional domain that in other enzymes recognizes linear peptides. We characterize the activity of the enzyme on two substrates: one with a peptide leader and one without. Kinetics and biophysics reveal that the leader on the substrate is not recognized by the enzyme. The enzyme is faster on either substrate than the macrocyclase or protease in vitro. The enzyme has a preferred order of oxidation of multiple thiazolines in the same linear peptide.

Insights into the Mechanism of the Cyanobactin Heterocyclase Enzyme

“…7 The “full length” substrate peptides PatE2K and PatE3KK (Scheme S4) were expressed with a C-terminal, noncleavable his-tag and purified as previously described. 19…”

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

“…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

“…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.…”