Objective
The recent cloning and sequencing of the monensin biosynthetic gene cluster has opened up new opportunities for exploring the mechanism of chain synthesis and oxidative cyclisation. The aim of this project is to study the enzymology of the initial modules of the monensin polyketide synthase (PKS) and thus help propose structures for the biosynthetic intermediates.
Two complementary approaches will be used:
i) chemical synthesis of labelled analogues of the proposed intermediates for in viro and in vitro feeding studies and;
ii) genetic manipulations of the monensin cluster in order to probe for partially processed intermediates on the pathway.
Such intermediates would provide specific substrates for direct assay of purified recombinant PKS and of the enzymes that are responsible for oxidative cyclisation. Enantiomerically pure labeled di-, tri- and tetraketide units, analogues of the proposed interrnediates after module 1, 2 and 3 of the monensin PKS respectively , will be synthesised, and used for in vivo feeding studies. Their fate will be monitored by isolation of labelled metabolites and characterisation by using NMR or mass spectrometry . Meanwhile, truncated versions of the monensin PKS multienzyme will be constructed containing either the first 1, 2, 3 or 4 modules ofthe monensin A-producing PKS. The covalent C-terminal attachment of a thioesterase that catalyses chain release from similar PKSs will allow the truncated polyketide to be released from the enzyme for identification The results should reveal the intriguing enzymology of oxidative cvclisations, and clearly show the relationship between polyether and macrolide biosynthesis.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- medical and health sciences medical biotechnology genetic engineering
- natural sciences chemical sciences analytical chemistry mass spectrometry
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
CB2 1GA CAMBRIDGE
United Kingdom
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