Chemoenzymatic synthesis of thiazolyl peptide natural products featuring an enzyme-catalyzed formal [4 + 2] cycloaddition

Abstract

Thiocillins from Bacillus cereus ATCC 14579 are members of the well-known thiazolyl peptide class of natural product antibiotics, the biosynthesis of which has recently been shown to proceed via post-translational modification of ribosomally encoded precursor peptides. It has long been hypothesized that the final step of thiazolyl peptide biosynthesis involves a formal [4 + 2] cycloaddition between two dehydroalanines, a unique transformation that had eluded enzymatic characterization. Here we demonstrate that TclM, a single enzyme from the thiocillin biosynthetic pathway, catalyzes this transformation. To facilitate characterization of this new class of enzyme, we have developed a combined chemical and biological route to the complex peptide substrate, relying on chemical synthesis of a modified C-terminal fragment and coupling to a 38-residue leader peptide by means of native chemical ligation (NCL). This strategy, combined with active enzyme, provides a new chemoenzymatic route to this promising class of antibiotics.

Figure 1

(a) Biosynthesis of thiocillins from a modified peptide. LP = leader peptide. (b) Thiocillin gene cluster from Bacillus cereus ATCC 14579. Open reading frames encoding key post-translational enzymes are shown. (c) Sequence of the 52-residue precursor featuring a 38-residue leader sequence (gray) and a 14-residue core peptide (black).

Figure 2

(a) Synthesis of the TclM substrate by thiophenol-catalyzed native chemical ligation and sulfhydryl elimination with dibromoadipamide. (b, c) QTOF-MS data of (b) NCL product 15 and (c) elimination product 16.

Figure 3

(a) SDS-PAGE of purified recombinant TclM. (b) Overlaid extracted ion chromatograms of TclM substrate and TclM assay after 20 h of incubation. The labeled peak indicates the newly formed thiazolyl peptide. (c) MS/MS spectrum of the isolated artificial thiazolyl peptide prepared by the chemoenzymatic method. Ions are labeled according to convention for the cyclic peptide. (d) Aromatic region of the ¹H NMR spectrum showing new pyridine protons.

Figure 4

Proposed mechanism of formal [4 + 2] cycloaddition catalyzed by TclM. The reaction may proceed in concerted fashion from the imidic acid, as illustrated, or else stepwise (not shown) to yield putative intermediate I. This undergoes further elimination/aromatization to the trithiazolylpyridine core of the thiocillins (structure III, R1, R2 = thiazole).