Substrate analogue studies on cephalosporin biosynthesis enzymes

The application of β-lactams as chemotherapeutic agents in the 1940s fundamentally changed the course of medicine. Some 80 years later, β-lactam antibiotics still have a central role in modern medicine. The biosynthesis of β-lactams is arguably as remarkable as their applications. The research described in this thesis focussed on the first two steps in the biosynthesis of cephalosporins from penicillin N; the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the subsequent 3’-hydroxylation of DAOC to deacetylcephalosporin C (DAC). These two steps are catalysed by a single enzyme (DAOCS/DACS) in eukaryotic fungi (A. chrysogenum), and by two distinct enzymes (DAOCS and DACS) in prokaryotes (S. clavuligerus). These three enzymes are notable both from mechanistic and medicinal perspectives.
Chapter 2 describes updated expression protocols for the production of recombinant DAOCS/DACS; the DAOCS/DACS gene (cefEF) has historically proved recalcitrant to soluble expression in E. coli. Through the application of modern recombinant expression techniques, such as codon-optimisation and the use of a pCold-derived expression vector, highly active DAOCS/DACS was afforded in pleasing yield and purity. DAOCS/DACS was found to be active under simplified assay conditions compared with previous studies, and was stable after several freeze-thaw cycles. These modified expression conditions for cefEF provided a resilient stock of DAOCS/DACS with which mechanistic inquiries could be directed. Procedures for producing recombinant DAOCS and DACS were also developed.
Chapter 3 describes investigations into the hydroxylase activity of DAOCS/DACS using the 3-vinyl cephem substrate analogue. Incubations of the 3-vinyl cephem with DAOCS/DACS produced a diol under standard assay conditions. When the incubations were carried out with addition of 5 mM sodium azide, the product was a cephem hydroxy azide which was isolated by HPLC and robustly characterised by LC-MS and NMR. It is proposed that azide insertion is catalysed by DAOCS/DACS, in which case this would be the first reported enzyme-catalysed incorporation of the azide functional group into the cephalosporin scaffold. DACS was also shown to catalyse the azidation reaction. Derivatisation of the azide functional group may be a route to new cephalosporin antibiotics. More generally, DAOCS/DACS-catalysed azidation suggests that the ability of 2OG oxygenases to catalyse azidation is more widespread than previously thought; the insertion of the azide group into other 2OG oxygenase substrates (e.g., lipids and proteins) should be investigated. In Chapter 4, attempts to crystallise DAOCS/DACS, as well as a mutant of DAOCS (ICM DAOCS) are described. A new co-crystallisation method, coupled with seeding experiments, produced encouraging crystalline precipitates with DAOCS/DACS and ICM DAOCS. Further optimisation is required to yield macrocrystals capable of diffraction. This work provides a foundation for the structural characterisation of DAOCS/DACS, DAOCS, and DACS. Investigations into the 3’-hydroxylation of DAOC to DAC by 18O-labelling experiments are also described in Chapter 4. The results confirm previous observations of incorporation of oxygen from water as well as from O2 into the hydroxyl group of DAC. By contrast, conversion of the 3-vinyl cephem analogue to give the hydroxy azide products proceeds without incorporation of water derived oxygen; in the case of the diol, one oxygen comes from water and one from O2.
Overall, as described in Chapter 5, the work furthers the mechanistic understanding of the hydroxylase activities of DAOCS/DACS and DACS. The results imply they catalyse oxidative transformations by at least two distinct mechanisms, depending on the structure of substrate being converted. It is hypothesised that a HO−Fe(IV)=O species, with the hydroxyl ligand derived from a water molecule, could account for the observed partial incorporation of oxygen from water into some, but not all products. Such an intermediate may be involved in catalysis in other 2OG oxygenases, including those involved in clavam biosynthesis and certain human oxygenases.

Keywords: Substrate analogoues
Antibiotics
Beta-lactams
Cephalosporins
Advisor: Schofield, C