Objective
Penicillin and cephalosporin antibiotics are amongst the most important therapeutic agents in use (est.
size of antibiotic market is 15 billion ECU per year). Some penicillins are produced by direct fermentation, but all cephalosporin and most penicillin antibiotics are produced by complex and expensive synthetic modifications of fermented products. The objective of this project is to develop our understanding of the structures and mechanism of two enzymes which catalyse key steps in penicillin and cephalosporin biosynthesis to allow engineering of their specificity. Subsequently, the modified enzymes will be introduced into micro-organisms so that penicillins and cephalosporins of choice may be directly fermented without the need for synthetic modification, with concomitant reductions in production costs and toxic by-products. The key steps in penicillin and cephalosporin biosynthesis are catalysed by the ferrous dependent oxygenases, isopenicillin N synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), respectively. The genes for both of these enzymes have been identified, cloned and over expressed. The crystal structure of an inactive form of IPNS has been solved by us and the structure determination of DAOCS is in progress. In order to identify the key residues for catalysis and substrate specificity structural data on enzyme substrate/intermediate/product complexes is required. This will involve the development of new techniques for fast protein purification, anaerobic co-crystallization of these highly labile and oxygen sensitive enzymes and for the triggering and monitoring of reactions in the crystalline state. The latter will include a combination of time-resolved diffraction techniques and other biophysical methods (e.g. time resolved single crystal X-ray absorption studies, mass spectrometric and electron spin resonance studies).
The results of the structural studies will be used to engineer the active sites of IPNS and DAOCS with the respective objectives of producing novel ß-lactam antibiotics, and cephalosporins with medicinally useful hydrophobic side chains. Engineered DAOCS will also be used in attempts to produce 3- exomethylene cephalosporin C, the key intermediate in the production of the most valuable cephalosporins. Engineered IPNS and DAOCS will be screened in kinetic and substrate specificity analyses in order to evaluate the suitability of the modified enzymes for the desired functions.
There is intense scientific, medicinal and commercial interest in other members of the family of ferrous dependent oxygenases to which IPNS and DAOCS belong, e.g. prolyl hydroxylase, which is involved in collagen biosynthesis and l-aminocyclopropane- l- carboxylate oxidase (ACCO, which catalyses the key step in the biosynthesis of the plant hormone, ethylene) are targets for inhibition; and clavaminic acid synthase which is involved in the biosynthesis of the most important ß-lactamase inhibitor, clavulanic acid, are all members of this family. The only structural studies reported so far are those on IPNS. Thus, this study is likely to have an impact very much wider than its immediate objectives and may have far reaching commercial implicahons. The sequential priorities of the work programme directly flow from the integrated objectives of the proposal, which is designed to fulfil the research tasks listed under Area 6 (Structural Biology) in the Biotechnology 1994-1998 Programme.
Our objectives are i) to develop fast protein purification and anaerobic crystallization techniques, ii) to develop techniques for triggering and monitoring reactions in crystals, iii) to continue the development of fast data collections methods, iv) to apply time-resolved X-ray diffraction techniques in order to determine the three dimensional structures of
substrate/intermediate/product complexes of IPNS and DAOCS, and v) in the light of these results, to introduce new catalytic activities into IPNS and DAOCS by protein engineering.
It is anticipated that once these objectives are achieved application of the results to the development of "factory cells" for the new and improved routes to antibiotics will form the basis of a separate proposal, in conjunction with pharmaceutical companies. We expect to begin exploratory work along these lines during the final year of the proposed project.
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.
- natural sciences earth and related environmental sciences geology mineralogy crystallography
- medical and health sciences basic medicine pharmacology and pharmacy pharmaceutical drugs antibiotics
- natural sciences chemical sciences organic chemistry aliphatic compounds
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
- natural sciences biological sciences molecular biology structural biology
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Coordinator
OX1 3QT OXFORD
United Kingdom
The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.