Final Activity Report Summary - MEGOTAB (Mechanism and exploitation of glycosyltransferases involved in antibiotic biosynthesis)
The majority of antibiotic substances in use today are natural products that certain bacteria and fungi produce and export from the cell. Sugars are common constituents of these antibiotics and are incorporated into the natural product by specific enzymes known as glycosyltransferases. Over the last decade there has been a global emergence of antibiotic resistance and, at the same time, no new antibiotics have been discovered. Therefore it has become particularly important to modify the pre-existing ones to improve their potency. A very promising strategy towards this consists in the alteration or addition of sugars using glycosyltransferases in vitro.
In this research project we aimed to investigate the final steps of the biosynthesis of teicoplanin by a. Teichomyceticus. Along with vancomycin, teicoplanin is a glycopeptide antibiotic of last resort for the treatment of widespread and life-threatening infections such as the methicillin-resistant staphylococcus aureus (MRSA). In particular, we were interested in a new type of bacterial glycosyltransferase that allegedly attached the sugar d-mannose to teicoplanin. The mannosylation of natural products is a rare event and its mechanism was still uncertain. Furthermore, we envisaged that the mannosyltransferase could be used to generate a range of new compounds of potentially high medicinal interest.
The gene tcp orf3*, encoding for the teicoplanin mannosyltransferase, was cloned from the deoxyribonucleic acid (DNA) of a.teichomycetus and, based on this, we were able to express the enzyme in e. coli as host. From its sequence we foresaw that the enzyme could be an integral membrane protein, therefore difficult to isolate and handle. As such it was found to be located in soluble e. coli membranes that we were able to isolate by ultracentrifugation for in vitro testing. This last task was still in progress by the time of the project completion due to the initial unavailability of the specific d-mannose donors required by the enzyme. These donors were thought to be mannosylated phospholipids of complex composition, whose chemical preparation was challenging. For these reasons we chose to prepare them biosynthetically, through exploitation of the action of another recently identified bacterial glycosyltrasferase known as dolichol-phospho-beta-d-mannose synthase (DPMS). The gene SC6D7.16 was cloned from the DNA of s. coelicolor A(3)2 and, from it, the DPMS was successfully expressed in soluble form in e. Coli, purified and characterised. The DPMS was found to selectively employ guanosine diphosphate (GPD) mannose along with commercially available dolichylphospholipids to produce beta-d-mannosyldolichylphospholipids, currently being assessed as substrates for the teicoplanin mannosyltransferase.
Despite some modifications of the original research plan and the fact that no novel glycopeptides were generated yet, the fellows work established the ground for further studies on the biology of the bacterial glycosyltransferases and for their utilisation in the preparation of pharmaceuticals. Moreover, because of the requirements and spin-offs of this project, new significant research objectives were identified and targeted, such as the structural characterisation and utilisation of the dolichol-phospho- beta -d-mannose synthase, which was a key enzyme for human and pathogenic glycosylation, and the elucidation of the d-mannose function in teicoplanin antibiotic action.
Combating and curing major diseases, apart from improving the quality of healthcare, was one of the social and scientific priorities of the European Community as expressed by the Sixth Framework Programme. By addressing the emerging resistance to last-resort antibiotics the outcomes of this project would hopefully contribute to the benefit of both scientific and non-scientific communities.
In this research project we aimed to investigate the final steps of the biosynthesis of teicoplanin by a. Teichomyceticus. Along with vancomycin, teicoplanin is a glycopeptide antibiotic of last resort for the treatment of widespread and life-threatening infections such as the methicillin-resistant staphylococcus aureus (MRSA). In particular, we were interested in a new type of bacterial glycosyltransferase that allegedly attached the sugar d-mannose to teicoplanin. The mannosylation of natural products is a rare event and its mechanism was still uncertain. Furthermore, we envisaged that the mannosyltransferase could be used to generate a range of new compounds of potentially high medicinal interest.
The gene tcp orf3*, encoding for the teicoplanin mannosyltransferase, was cloned from the deoxyribonucleic acid (DNA) of a.teichomycetus and, based on this, we were able to express the enzyme in e. coli as host. From its sequence we foresaw that the enzyme could be an integral membrane protein, therefore difficult to isolate and handle. As such it was found to be located in soluble e. coli membranes that we were able to isolate by ultracentrifugation for in vitro testing. This last task was still in progress by the time of the project completion due to the initial unavailability of the specific d-mannose donors required by the enzyme. These donors were thought to be mannosylated phospholipids of complex composition, whose chemical preparation was challenging. For these reasons we chose to prepare them biosynthetically, through exploitation of the action of another recently identified bacterial glycosyltrasferase known as dolichol-phospho-beta-d-mannose synthase (DPMS). The gene SC6D7.16 was cloned from the DNA of s. coelicolor A(3)2 and, from it, the DPMS was successfully expressed in soluble form in e. Coli, purified and characterised. The DPMS was found to selectively employ guanosine diphosphate (GPD) mannose along with commercially available dolichylphospholipids to produce beta-d-mannosyldolichylphospholipids, currently being assessed as substrates for the teicoplanin mannosyltransferase.
Despite some modifications of the original research plan and the fact that no novel glycopeptides were generated yet, the fellows work established the ground for further studies on the biology of the bacterial glycosyltransferases and for their utilisation in the preparation of pharmaceuticals. Moreover, because of the requirements and spin-offs of this project, new significant research objectives were identified and targeted, such as the structural characterisation and utilisation of the dolichol-phospho- beta -d-mannose synthase, which was a key enzyme for human and pathogenic glycosylation, and the elucidation of the d-mannose function in teicoplanin antibiotic action.
Combating and curing major diseases, apart from improving the quality of healthcare, was one of the social and scientific priorities of the European Community as expressed by the Sixth Framework Programme. By addressing the emerging resistance to last-resort antibiotics the outcomes of this project would hopefully contribute to the benefit of both scientific and non-scientific communities.