Mechanistic Studies on the Remarkable Epimerisations of Clavam and Carbapenem Biosynthesis

Despite problems of resistance, antibiotics are arguably the most important molecules used in medicine. Worldwide the most important antibiotics, by commercial or medicinal standards, are the penicillins and related structures all of which contain a four membered beta-lactam ring. The beta-lactam characterises not only the penicillins but also other important antibiotics such as the cephalosporins. This ring is vital for antibacterial activity since it reacts with the enzymes located in the bacterial cell wall that are the targets of these antibiotics. When the antibiotics neutralise their target enzymes the bacteria cannot properly synthesise their cell walls and so cannot survive. As Fleming observed, despite the observations that penicillins are lethal to many bacteria, beta-lactams are actually produced by microorganisms. In fact the structures of the naturally occurring bicyclic beta-lactam antibiotics are so complex and unusual that it is highly improbable they would be discovered by human synthesis either by design or chance. With few exceptions all the beta-lactams in use are produced either by direct fermentation of microorganisms or by synthetic modification of fermented materials, as their total synthesis from petrochemicals is too expensive. As with all antibiotic families the continued use of beta-lactams is threatened by resistance. This can take various forms including camouflage of the target and the evolution of molecular pumps to expel the antibiotics. One important resistance mechanism involves enzymes that break the beta-lactam ring by addition of water. These beta-lactamases have evolved to be highly efficient as their activity can be a matter of life or death for bacteria. To counter the activity of beta-lactamases humans have developed new families of antibiotics that are less susceptible to beta-lactamase mediated hydrolysis or actually inhibit beta-lactmase activity. Such families include the cephalosporins and the carbapenems. Remarkably the clinically useful inhibitors have themselves all turned out to be beta-lactams. Some of these compounds were very potent beta-lactamase inhibitors but were not powerful enough antibiotics for sole use, so are formulated with a penicillin antibiotic. There is an ongoing need for new antibiotics and beta-lactamase inhibitors as bacteria continually evolve better methods of resistance. A limitation in this development is the production costs of useful compounds. In the case of the carbapenems this problem is particularly acute as no method for their commercially viable fermentation has been developed, so they are prepared by expensive total synthesis. We have been studying the routes by which microorganisms make beta-lactams. Our work together with that of others has revealed that bicyclic beta-lactams are produced by the action of chemically remarkable enzymes. The enzymes that catalyse the biosynthesis of the two rings of the four most important groups of beta-lactams, the penicillins, the cephalosporins, the clavams and the carbapenems, have been identified. In order to bind the antibiotics to their molecular targets these rings have to be both modified or functionalised with other chemical groups and their three dimensional shape (stereochemistry) has to be changed. In the new work we aim to attempt to understand how these unusual reactions, most of which are poorly understood, occur. The work is of practical significance in terms of developing new or more efficient routes to antibiotics; because the enzymes involved catalyse highly unusual reactions we envisage that the work will have unenvisaged applications. This was the case in work on the enzymes involved in the production of the beta-lactam rings which has turned out to have widespread implications for work on the mechanism by which animal cells respond to low oxygen concentrations and has applications in cancer and heart disease.

We have successfully defined the structures and reactions of the enzymes catalysing the biosynthesis of the nuclei of the penicillins, cephalosporins, and clavams. Further, enzymes for the biosynthesis of (5R)-carbapenem-3-carboxylate, were partially characterised. After the beta-lactam nuclei are formed they undergo chemically unusual transformations, including striking epimerisations during carbapenem/clavam biosynthesis. We request funding to identify/analyse the enzymes catalysing these unusual processes focusing on the remarkable (5S)- to (5R)-epimerisations in carbapenem and clavam biosynthesis. The aims are ambitious and there are considerable challenges, not least in functional assignments on the clavam pathway, where the intermediates are labile/synthetically inaccessible, but we have extensive expertise in the field. An approach employing techniques from chemistry, biochemistry, cell biology and structural biology will be used. We request funding for 2 PDRAs plus technical support. One PDRA will focus on synthesis of (labelled) intermediates, recombinant protein production and in vitro characterisation and the other on work with S. clavuligerus (including immunoprecipitation, two hybrid and affinity purification methods) and biophysical (including EPR and crystallographic) analyses). Technical milestones are well-defined and include: (i) production of enzymes involved in the epimerisations in soluble recombinant form with the generation of antibodies; (ii) definition of the stereochemical/mechanistic course of the epimerisations using labelled substrates prepared by chemo-enzymatic routes; (iii) biophysical analyses for the target enzymes complexed with substrates/intermediates (incl. crystallography and EPR); (iv) functional assignments and mechanistic investigations of the proteins mediating epimerisation by the use of new techniques (where required) combining in vitro and vivo analyses employing labelled substrates.