Enzymatic basis of ribosomal peptide prenylation in cyanobacteria - PubMed (original) (raw)

. 2011 Aug 31;133(34):13698-705.

doi: 10.1021/ja205458h. Epub 2011 Aug 4.

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Enzymatic basis of ribosomal peptide prenylation in cyanobacteria

John A McIntosh et al. J Am Chem Soc. 2011.

Abstract

The enzymatic basis of ribosomal peptide natural product prenylation has not been reported. Here, we characterize a prenyltransferase, LynF, from the TruF enzyme family. LynF is the first characterized representative of the TruF protein family, which is responsible for both reverse- and forward-O-prenylation of tyrosine, serine, and threonine in cyclic peptides known as cyanobactins. We show that LynF reverse O-prenylates tyrosine in macrocyclic peptides. Based upon these results, we propose that the TruF family prenylates mature cyclic peptides, from which the leader sequence and other enzyme recognition elements have been excised. This differs from the common model of ribosomal peptide biosynthesis, in which a leader sequence is required to direct post-translational modifications. In addition, we find that reverse O-prenylated tyrosine derivatives undergo a facile Claisen rearrangement at 'physiological' temperature in aqueous buffers, leading to forward C-prenylated products. Although the Claisen rearrangement route to natural products has been chemically anticipated for at least 40 years, it has not been demonstrated as a route to prenylated natural products. Here, we show that the Claisen rearrangement drives phenolic C-prenylation in at least one case, suggesting that this route should be reconsidered as a mechanism for the biosynthesis of prenylated phenolic compounds.

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Figures

Figure 1

Figure 1. Prenylation in natural products

(A) Representative cyanobactin peptide natural products, showing groups derived from DMAPP in blue. (B) Two possible enzymatic mechanisms of phenol _ortho_-C-prenylation. First, DMAPP is dephosphorylated to yield a cation that can react either at oxygen (pathway I) or at carbon (pathyway II). In principle, a Claisen rearrangement from pathway I could then yield the C-prenylated product. Only pathway II has been previously linked to enzymatic modification.

Figure 2

Figure 2. Defining the biosynthetic route to prenylated cyanobactins

Proposed biosynthetic scheme for lyn pathway showing modification of precursor peptide by heterocyclization, proteolysis, macrocyclization and prenylation. The first steps in this route are supported by previous enzymological studies, but the timing of prenylation was not known. Enzyme recognition elements are highlighted. Analogues (3–6) were used to assess prenylation of each possible biosynthetic intermediate and are shown in analogous colors beneath each proposed biosynthetic intermediate. Pro was substituted as an approximate isostere for thiazole in later analogues. No reaction was observed with any intermediate except the final cyclic peptide, showing that prenylation occurs at a late step, after all enzyme recognition elements have been excised.

Figure 3

Figure 3. LynF is _ortho_-C-prenylating

The aromatic / olefin region of the HSQC NMR spectra are shown for boc-tyrosine (14) (A) and its purified enzymatic product with LynF (B), clearly indicating a single, forward C-prenylation event. Similar spectra were also observed for reactions containing 6. NMR and MS characterization of compounds is presented in Supporting Figures S1, S3, and S5.

Figure 4

Figure 4. LynF catalyzes reverse O-prenylation of tyrosine

The aromatic / olefin region of 1H NMR spectra are shown (A) for boc-tyrosine (14) (B) the HPLC-purified intermediate LynF product (31), and (C) the final reaction product (30). These spectra clearly indicate that the first product of the LynF reaction is reverse-O-prenyl tyrosine, which subsequently rearranges to give the C-prenylated product.

Figure 5

Figure 5. Time course of boc-L-Tyr (14) reaction followed by HPLC

The reaction was performed in quadruplicate, with variation indicated by error bars (A), (B). Initial product of the reaction is almost exclusively O-prenylated as shown at 1 h, 2 h, and 4 h time points (B). After 4 h, the level of O-prenylated intermediate reaches steady-state and its levels are constant through 24 h, accompanied by steady increase in the concentration of C-prenylated final product 29. This allowed kinetic constants for the Claisen rearrangement to be directly determined, since at steady state the concentration of the O-prenyl intermediate can be assumed to be a constant.

Figure 6

Figure 6. Phylogenetic analysis of the TruF / LynF family

(A) TruF-group proteins cluster according to whether natural products are prenylated (top) or non-prenylated (bottom). Chemical products are shown for each TruF-like protein, where they exist. No TruF-like sequence relatives can be identified outside of cyanobactin gene clusters using either BLAST searching or sequence alignments with other PT family proteins, indicating that this is a novel group of PTs. (B) Actual product structures of prenylagaramide (left) and trunkamide (right) pathways; predicted enzymatic product of lyn pathway (middle) prior to Claisen rearrangement.

Scheme 1

Scheme 1. NMR characterized products

Scheme 2

Scheme 2. Claisen rearrangement pathway

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