Using marine natural products to discover a protease that catalyzes peptide macrocyclization of diverse substrates - PubMed (original) (raw)

Using marine natural products to discover a protease that catalyzes peptide macrocyclization of diverse substrates

Jaeheon Lee et al. J Am Chem Soc. 2009.

Abstract

Small N-C terminally cyclic ribosomal peptides are common in nature, yet the mechanisms underlying their biosyntheses remain largely unknown. We recently identified candidate peptide cyclase genes in the metagenomes of marine animals. Here we report that PatG, a protease discovered in this analysis, cleaves variables, short peptides out of a precursor protein, and cyclizes these peptides in vivo and in vitro.

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Figures

Figure 1

Figure 1

N-C cyclic peptides of the cyanobactin group. The partial precursor peptide sequences are shown that give rise to the cyclic products. Amide bonds formed by PatG and its relatives are shown in green, while recognition sequences are shown in bold and cassette sequences leading to products are underlined. Patellin 2, ulithiacyclamide, and trichamide are natural products that require multiple enzymatic processing steps in their synthesis, while the eptidemnamide precursor was engineered and requires only cleavage and cyclization.

Figure 2

Figure 2. PatA catalyzes two N-terminal cleavage reactions

(a) The precursor peptide PatEdm is cleaved by PatA at a recognition sequence to release first (5) and then (4), with free N-termini for cyclization. (b) SDS-PAGE of a time-course experiment with PatA and PatEdm shows that the first site is slowly cleaved, followed by relatively rapid proteolysis of the second site. (c) All reaction products were observed by mass spectrometry

Figure 3

Figure 3. PatG catalyzes cyclization by a transamidation mechanism

(a) Starting from product (5) of PatA, PatG cleaves this peptide into a cyclic compound, eptidemnamide, and a small linear fragment representing the recognition sequence. (b) High resolution mass spectrometry and comparison to authentic standards confirmed that this cyclic peptide was formed from compound (5).

Figure 4

Figure 4. Proposed transamidation mechanism of peptide cyclization

In this simplified scheme, the active-site Ser of PatG is shown forming an enzyme-substrate tetrahedral intermediate with loss of the AYDGE recognition sequence. Subsequently, the N-terminus of the substrate attacks the activated ester bond to release a cyclic peptide and the free enzyme.

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