Marine molecular machines: heterocyclization in cyanobactin biosynthesis - PubMed (original) (raw)

Marine molecular machines: heterocyclization in cyanobactin biosynthesis

John A McIntosh et al. Chembiochem. 2010.

Abstract

Natural products that contain amino-acid-derived (Cys, Ser, Thr) heterocycles are ubiquitous in nature, yet key aspects of their biosynthesis remain undefined. Cyanobactins are heterocyclic ribosomal peptide natural products from cyanobacteria, including symbiotic bacteria living with marine ascidians. In contrast to other ribosomal peptide heterocyclases that have been studied, the cyanobactin heterocyclase is a single protein that does not require an oxidase enzyme. Using this simplifying condition, we provide new evidence to support the hypothesis that these enzymes are molecular machines that use ATP in a product binding or orientation cycle. Further, we show that both protease inhibitors and ATP analogues inhibit heterocyclization and define the order of biochemical steps in the cyanobactin biosynthetic pathway. The cyanobactin pathway enzymes, PatD and TruD, are thiazoline and oxazoline synthetases.

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Figures

Figure 1

Figure 1

Heterocyclic natural products. Shown are patellamide C (1), epothilone B (2), thiostrepton (3), and microcin B17 (4).

Figure 2

Figure 2

A) Shown are alignments between PatD and TruD. Darker regions indicate regions of higher identity. B) Sequence of TruE2 precursor peptide is shown, with naturally heterocyclized residues highlighted in red. C) A zoomed-in view of the C-terminal cassette in TruE2. In vitro, PatD modifies one Thr and one Cys in this cassette, while TruD modifies one Cys both in vitro and in vivo. In nature, in combination with other biosynthetic enzymes the TruD product shown is converted to the prenylated, heterocyclic natural product patellin 6.

Figure 3

Figure 3

Stoichiometry of heterocycle formation. A) rates of ADP formation and thiazoline synthesis are overlaid; corrected slope denotes the rate of ATP hydrolysis when corrected for the background hydrolysis. B) %-completion of the heterocyclization reaction as determined by SDS-PAGE gel densitometry. C) SDS-PAGE gel used to derive [thiazoline] and %-completion.

Scheme 1

Scheme 1

Shown above are mechanistic possibilities for heterocyclization. A) oxidation preceding heterocycle formation B) activation of the adjacent carbonyl oxygen, perhaps with phosphate from ATP C) intein mechanism for thiazoline formation as a side-product D) molecular machine mechanism for heterocyclase enzymes.

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