Mechanistic Studies on the Remarkable Epimerisations of Clavam and Carbapenem Biosynthesis

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

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.

Technical Summary

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.

Publications


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Batchelar ET (2008) Thioester hydrolysis and C-C bond formation by carboxymethylproline synthase from the crotonase superfamily. in Angewandte Chemie (International ed. in English)
Caines ME (2009) Structural and mechanistic studies on N(2)-(2-carboxyethyl)arginine synthase. in Biochemical and biophysical research communications
Gruber T (2012) 3-Meth-oxy-3-oxopropanaminium chloride. in Acta crystallographica. Section E, Structure reports online
Gruber T (2012) Methyl 6-amino-6-oxohexa-noate. in Acta crystallographica. Section E, Structure reports online
Hamed RB (2010) Carboxymethylproline synthase catalysed syntheses of functionalised N-heterocycles. in Chemical communications (Cambridge, England)
Hamed RB (2016) Use of Methylmalonyl-CoA Epimerase in Enhancing Crotonase Stereoselectivity. in Chembiochem : a European journal of chemical biology
 
Description Our project aimed to develop an understanding of how the ring structures of some of the most important antibiotics in current use are constructed and modified in microorganisms. The work built upon studies that have defined the structures and mechanisms of the enzymes catalysing the biosynthesis of the nuclei of the famous penicillins and the related cephalosporins. The key common functional element in these antibiotics is the beta-lactam ring, modification of which determines not only the spectrum of activity but also susceptibility to antibiotic destroying enzymes, or beta-lactamases. We have now analysed how Nature constructs and, as a focus of the current work, modifies the rings of the carabapenem antibiotics, which are sometimes used as 'last resort' treatment antibiotics and of clavulanic acid, which although it contains a beta-lactam ring, acts as a 'guardian angel' for beta-lactam antibiotics by neutralising beta-lactamases. One productive area of work has been to investigate the mechanism by Nature modifies the activity of the rings by 'switching' the three-dimensional of atoms that make them (epimerisation). We have carried out extensive studies on the key epimerase involved in carbapenem biosynthesis and furthermore shown that the same mechanism is conserved in different species. We have found that variation of the same ring framework can be achieved not only by modification of the ring system once constructed but also by varying the staring material for producing the rings. Work on the first ring forming enzyme in the carbapenem biosynthesis pathway has led us to pioneer studies on the application of a superfamily of enzymes (the crotonases) for the stereoselective synthesis of functionalised rings containing nitrogen not only leading to beta-lactams but also to other ring containing nitrogen of interest to the pharmaceutical industry. This work has attracted attention both in the synthetic chemistry and biochemistry communities. It has led to the development of new 'semisynthetic' procedures (i.e. involving both biocatalysts and synthesis) for the preparation of highly functionalised bicyclic beta-lactams including, by protein engineering, of those that were hitherto inaccessible due to the density of functionalisation. The work has involved considerable efforts to develop methodology for functional assignments of enzymes with labile small molecule substrates with a focus being on techniques that couple liquid chromatography and mass spectrometric analyses. Some of the methods are generally useful and have been applied to human proteins. Structural analyses were an integral part of the work and were used to guide the functional analyses. We have reported > 10 new structures of enzymes involved in the biosynthetic pathways including some reflecting intermediate complexes. From a mechanistic perspective highlights included studies on enzymes proceeding via acyl-enzyme complexes, oxygenase-epimerases and on carboxymethylproline synthases. The work has resulted in multiple publications, some in high profile journals and a patent application. Aside from basic advances in our understanding of how nature constructs complex and labile molecules of biomedicinal importance, we feel that a highlight has been to reveal the potential of the crotonase superfamily enzymes as versatile and useful enzymes for the production of a range of ring structures of pharmaceutical interest in the antibiotic field and beyond.
Exploitation Route Perhaps the most important outcome of the work has been to open up the crotonase enzyme superfamily for use as biocatalysts. To date the crotonase superfamily has been neglected in this regard. It has the potential to enable to efficient production of many small-molecules of interest to the pharmaceutical and agrochemical industries.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description One important outcome of our work has been to pioneer the use of crotonase superfamily enzymes for the biocatalytic production of N-heterocycles in very high disteroselectivity. This work reported in Nature Chemistry, and which is the subject of a patent application, opens the way for engineering of the ubiquitous crotonase superfamily to produce pharmaceutically useful heterocycles, which otherwise are only available by expensive and environmentally unfriendly synthesis, by biocatalysis. We are presently writing up for publication the results of further studies which demonstrate the potential of the methodology to make highly functionalised bicylic ring structures in a stereoselective manner. These studies have been recognised both in the synthetic chemistry and biochemistry communities (see e.g. B Lust, JW Lee, Synfacts 2011, 6, 0679). As part of this work we have generated > 30 engineered carboxymethylproline synthases with defined activities in terms of making heterocycles; multiple new cell lines, liquid-chromatography-mass spectrometry and NMR based methods for analysing low molecular weight heterocycles in water, mass spectrometry based methodology for trapping enzyme intermediates, and NMR based methods for studying binding to paramagnetic metal centres in proteins.
First Year Of Impact 2011
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description 2nd World Epigenetics Summit, Munich, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at 2nd World Epigenetics Summit, Munich, Germany on "The role of oxygenases in epigenetics"
Year(s) Of Engagement Activity 2011
 
Description Chemistry & Biology Interface workshop, Salerno, Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at Chemistry & Biology Interface workshop, Salerno, Italy on "The molecular mechanism of oxygen sensing in humans"
Year(s) Of Engagement Activity 2011
 
Description Seminar at Sapienza University of Rome, Rome, Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at Seminar at Sapienza University of Rome, Rome, Italy on "The Chemical Basis of Oxygen Sensing in Humans"
Year(s) Of Engagement Activity 2011
 
Description Seminar at Vertex, Abingdon, Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation at Seminar at Vertex, Abingdon, Oxford on "Functional, Structural and Mechanistic Studies on Human Oxygenases
Year(s) Of Engagement Activity 2011