Antibiotic Biosynthesis

Most clinically used antibiotics are based upon natural products. The most important family of antibiotics contains a β-lactam ring, and includes the penicillin, cephalosporin, clavam, and carbapenem antibiotics. Our recent biosynthetic work has focused on the clavams and carbapenems, with a particular focus being on the mechanism and structures of enzymes that catalyse chemically 'interesting' steps. One of the clavams, clavulanic acid, is the most important clinically used β-lactamase inhibitor and like the penicillins and cephalosporins is produced by fermentation. This method has limited the production of modified clavams with improved clinical properties. In contrast the carbapenems are produced by total synthesis as it has not yet been possible to develop a viable commercially fermentation route to the clinically used antibiotics. We are interested in studying the biosynthetic pathways to the clavams and carbapenems with a view to developing routes to improved antibiotics and because the pathways involve some remarkable reactions from a chemical perspective.

Clavulanic Acid Mode of Action and Biosynthesis

β-Lactamases, which catalyse hydrolysis of the β-lactam ring, are amongst the most important mediators of antibiotic resistance and some β-lactams, including clavulanic acid, were explicitly developed as β-lactamase antagonists. Clavulanic acid inhibits Class A β-Lactamases by reacting to form acyl-enzyme complexes that are stable with respect to hydrolysis, a process closely related to the mode of action of β-lactam antibiotics such as the penicillins that inhibit transpeptidases involved in cell wall biosynthesis.

Although clavulanic acid is a small molecule (C8H8NO5) and contains only two chiral centres it is thermodynamically unstable and it has not been made via asymmetric total synthesis. We are investigating the biosynthetic pathway to clavulanic acid - our work involves structural work, functional analyses, and mechanistic studies. Techniques involved in this research include molecular biology, X-ray crystallography kinetics and organic synthesis. The latter is important since the late stage intermediates in the pathway are difficult to prepare and unstable. Studies on the multistep pathway (in collaboration with Inger Andersson and Janos Hajdu) have led to surprises including the trifunctional role of a single oxygenase and the fact that the pathway proceeds via intermediates that are almost enantiomers of the final product.

Recent work on the pathway has found proteins involved in the transport of intermediates and enzymes that catalyse reactions via acyl-enzyme intermediates.

Carbapenem Biosynthesis

The biosynthesis of carbapenem-5-carboxylate provides a model to understand the biosynthesis of the clinically useful carbapenem Thienamycin, an antibiotic more potent than most penicillins. In contrast to clavulanic acid biosynthesis, that of the simplest carbapenem requires only three steps - all of which we are presently investigating. The first enzyme CarB is an unusual member of the crotonase superfamily, the second CarA is a synthetase that in effect catalyses a reverse β-lactamase reaction, and the third CarC catalyses an unprecedented epimerisastion reaction.

In recent work we have been studying the structure and mechanism of CarB - a particular focus being on controlling the reactivity of the enolate intermediate in CarB catalysis. An ultimate goal would be enable the development of biocatalysts that can generate products of choice to react them with stereoselectivity with a range of electrophiles.

For reviews see:

Kershaw NJ, Caines MEC, Sleeman MC, Schofield CJ: The enzymology of clavam and carbapenem biosynthesis. Chem. Commun. 2005, 34: 4251-4263.
Hamed RB, Batchelar ET, Clifton IJ, Schofield CJ: Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity. Cell. Mol. Life Sci. 2008, 65: 2507-2527.
Baggaley KH, Brown AG, Schofield CJ: Chemistry and biosynthesis of clavulanic acid and other clavams. Nat Prod Rep 1997, 14: 309-333.

For representative publications see:

Hamed RB, Mecinović J, Ducho C, Claridge TDW, Schofield CJ: Carboxymethylproline synthase catalysed syntheses of functionalised N-heterocycles. Chem. Commun. (Camb.) 2010, 46: 1413-1415.
Mackenzie AK, Valegård K, Iqbal A, Caines MEC, Kershaw NJ, Jensen SE, Schofield CJ, Andersson I: Crystal Structures of an Oligopeptide-Binding Protein from the Biosynthetic Pathway of the [beta]-Lactamase Inhibitor Clavulanic Acid. Journal of Molecular Biology 2010, 396: 332-344.
Caines MEC, Sorensen JL, Schofield CJ: Structural and mechanistic studies on N2-(2-carboxyethyl)arginine synthase. Biochemical and Biophysical Research Communications 2009, 385: 512-517.
Cook KM, Hilton ST, Mecinović J, Motherwell WB, Figg WD, Schofield CJ: Epidithiodiketopiperazines Block the Interaction between Hypoxia-inducible Factor-1α (HIF-1α) and p300 by a Zinc Ejection Mechanism. Journal of Biological Chemistry 2009, 284: 26831 -26838.
Iqbal A, Clifton IJ, Bagonis M, Kershaw NJ, Domene C, Claridge TDW, Wharton CW, Schofield CJ: Anatomy of a Simple Acyl Intermediate in Enzyme Catalysis: Combined Biophysical and Modeling Studies on Ornithine Acetyl Transferase. Journal of the American Chemical Society 2009, 131: 749-757.
MacKenzie AK, Kershaw NJ, Hernandez H, Robinson CV, Schofield CJ, Andersson I: Clavulanic Acid Dehydrogenase:  Structural and Biochemical Analysis of the Final Step in the Biosynthesis of the β-Lactamase Inhibitor Clavulanic Acid†,‡. Biochemistry 2007, 46: 1523-1533.
Hamed RB, Batchelar ET, Mecinović J, Claridge TDW, Schofield CJ: Evidence that thienamycin biosynthesis proceeds via C-5 epimerization: ThnE catalyzes the formation of (2S,5S)-trans-carboxymethylproline. Chembiochem 2009, 10: 246-250.
Batchelar ET, Hamed RB, Ducho C, Claridge TDW, Edelmann MJ, Kessler B, Schofield CJ: Thioester hydrolysis and C-C bond formation by carboxymethylproline synthase from the crotonase superfamily. Angew. Chem. Int. Ed. Engl 2008, 47: 9322-9325.
Sorensen JL, Sleeman MC, Schofield CJ: Synthesis of deuterium labelled l- and d-glutamate semialdehydes and their evaluation as substrates for carboxymethylproline synthase (CarB)?implications for carbapenem biosynthesis. Chem. Commun. 2005, (9): 1155.
Sleeman MC, Sorensen JL, Batchelar ET, McDonough MA, Schofield CJ: Structural and Mechanistic Studies on Carboxymethylproline Synthase (CarB), a Unique Member of the Crotonase Superfamily Catalyzing the First Step in Carbapenem Biosynthesis. Journal of Biological Chemistry 2005, 280: 34956 -34965.
Elkins JM, Kershaw NJ, Schofield CJ: X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster. Biochem. J. 2005, 385: 565.
Topf M, Sandala GM, Smith DM, Schofield CJ, Easton CJ, Radom L: The Unusual Bifunctional Catalysis of Epimerization and Desaturation by Carbapenem Synthase. Journal of the American Chemical Society 2004, 126: 9932-9933.
Zhang Z, Ren J, Stammers DK, Baldwin JE, Harlos K, Schofield CJ: Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase. Nat. Struct. Biol 2000, 7: 127-133.
Valegård K, van Scheltinga AC, Lloyd MD, Hara T, Ramaswamy S, Perrakis A, Thompson A, Lee HJ, Baldwin JE, Schofield CJ, et al. Structure of a cephalosporin synthase. Nature 1998, 394: 805-809.