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Identification of a general biosynthetic pathway of 3-acyltetronates and further investigation of the biosynthetic pathway of the ionophoric polyethers tetronasin and tetronomycin

Final Report Summary - HAHN_PFL_TETRONATES (Identification of a general biosynthetic pathway of 3-acyltetronates and further investigation of the biosynthetic pathway of ionophoric polyethers ...)

Natural products are of continuing high interest both because of the fundamental insights and hints they offer us as to the course of evolution of life on earth; and as lead compounds in drug discovery. A key endeavour is to clarify the detailed mechanisms by which such molecules are assembled. One of the most important classes of bacterial natural products with diverse activities are polyketides. They are produced by so called Polyketide Synthases (PKSs), giant multienzymes which synthesize linear backbones by repetitive Claisen Condensations of malonyl monomers. The aim of this project was the elucidation of the biosynthesis of the near-mirror-image ionophoric polyether antibiotics tetronasin and tetronomycin by characterisation of key enzymes. Both natural products are produced by polyketide synthase-I (PKS-I) systems and are subject to a complex reorganisation of the molecular framework by non-PKS assembly line enzymes (tailoring enzymes) encoded in their gene clusters. These enzymes catalyse the formation of interesting functional groups, which are indispensable for the biological activity of the final natural products. In this project, the emphasis was on study of the biosynthesis of the tetronate and tetrahydrofuran rings.

Initial attempts to study the tetronate formation in the above-mentioned system failed because the candidate enzymes putatively responsible could not be expressed in a soluble form. It was therefore decided to switch to structurally simpler tetronate natural products. The phosphatase inhibitor RK-682 from Streptomyces sp. 88 682 represents a structurally simplified tetronate that was chosen to facilitate the analysis of the pathway's enzymology. Based on comparison to sequences of known gene products, the cloned RK-682 biosynthetic gene cluster was identified. Its identity was confirmed by a series of gene knockout experiments and heterologous expression of the whole cluster in Saccharopolyspora erythraea. The work on this project comprised in vitro reconstitution of parts of the pathway using recombinant enzymes and analysis of the results by HPLC MS. In the key experiment it was shown that the putative tetronate-forming enzyme RkD was indeed able to catalyse ring formation starting from acyl carrier protein (ACP)-bound 3-oxo-stearate and ACP-bound glycerate. These precursors were synthesized by Organic Chemistry. RkD shows high homology to FabH enzymes from fatty acid biosynthesis and is the first-ever reported tetronate forming enzyme. Homologous enzymes in the gene clusters of tetronasin and tetronomycin suggest a similar role of these enzymes within their biosynthesis.

In this project, the tailoring enzymology (late steps) within the biosynthetic pathways of tetronasin and tetronomycin was also studied. Both natural products share several functional groups, which are important for their biological activity. These groups are oxygen-containing heterocycles like tetrahydrofuran rings, tetrahydropyran rings and tetronate rings, but also one cyclohexyl ring. The heterocycles contribute significantly to the ability of these polyethers to complex cations, as part of their mechanism of action. Of particular importance for this project were the formation of the tetronate rings and the tetrahydrofuran rings. An interesting characteristic of both molecules is that they are constitutionally nearly identical, but are at the same time, perfect enantiomers in each and every one of their comparable stereo centres. Unlike most polyketides, neither molecule terminates in a carboxylic acid, but instead forms a tetronate ring. This suggests a unique release mechanism during their biosynthesis.

The epoxidases and epoxide hydrolases from the clusters of tetronasin and tetronomycin were cloned and expressed. While the expression of the B-enzymes was achieved relatively straightforwardly, the C-enzymes caused more problems. For TsnC, a reliable expression protocol could finally be developed. However, TmnC resisted all attempts to be expressed in soluble form. Therefore and because the only isolated intermediate stemmed from tetronasin biosynthesis, all following experiments were conducted for tetronasin. In order to closely mimic the PKS-bound state of the precursor, an ACP was cloned from the tetronasin gene cluster and heterologously expressed. Neither the cloning nor the expression proved to be problematic. The ACPs from the tetronasin-PKS are highly homologous, so the terminal ACP of module 13 was chosen as a representative. Substrate analogues of the natural polyketide chains were made by total synthesis and have for the first time made available plausible substrates for the key epoxidase enzyme TsnC. Meanwhile, X-ray crystallography of epoxide hydrolase/cyclase TsnB has led to the first-ever crystal structure for one of these usual enzymes.

This work provided valuable insights into the biosynthesis of tetronates and polyethers uncovering novel types of enzyme activity. This knowledge could be used in the future to manipulate biosynthesis of tetronates and polyethers and facilitate the development of novel, improved derivatives. For example, certain polyether natural products show striking selective toxicity towards cancer stem cells while sparing normal stem cells, and new derivatives of such compounds could help illuminate the mechanism of this effect and advance a novel therapy for cancer.

Contact details:

Prof. Peter F. Leadlay

Herchel Smith Professor of Biochemistry, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Old Addenbrookes Site, Cambridge CB2 1GA

Tel. + 44 1223-333656

Fax + 44 1223-766002.

E-Mail: pfl10@mole. bio. cam. ac. uk
http://www.bio. cam. ac. uk/~pflgroup/group. htm

Dr. Frank Hahn

Institute of Organic Chemistry

Leibnis University of Hanover

Room 116

Schneiderberg 1 B

30167 Hanover

Tel. + 49 511 762-4603

Fax + 49 511 762-3011

E-Mail: frank. hahn@oci. uni-hannover. de
http://uhw3dev. uni-hannover. de/oci/de/arbeitskreise/hahn/index. php