| Summary: | <p>This thesis describes an investigation of the mechanism of the bifunctional, a-ketoglutarate dependent dioxygenase, deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of this to deacetylcephalosporin C (DAC).</p> <p>The conversion of the unnatural substrate 3-exomethylene cephalosporin C by DAOC/DACS has been investigated in detail. A new metabolite was isolated from incubations of the deuterated [4-<sup>2</sup>H]-3-exomethylene cephalosporin C, and was identified as the 3β-spiroepoxide cepham, (2Ṟ,3Ṟ,6Ṟ,7Ṟ)-l-aza-[2-<sup>2</sup>H]-3-spiroepoxy-7-[(5Ṟ)-5-amino- 5-carboxypentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylic acid. The results obtained indicate that this metabolite is a shunt product whose formation is enhanced by the operation of a deuterium kinetic isotope effect on an enzyme-bound intermediate. It has also been found that this 3β-spiroepoxide cepham is further converted by DAOC/DACS to 3-formyl cephalosporoate products.</p> <p>The mechanism of oxygenation of DAOC/DACS was investigated through <sup>18</sup>O-labelling studies. Incubations of [2-<sup>13</sup>C,3-<sup>2</sup>H]penicillin N and [4-<sup>2</sup>H]-3-exomethylene cephalosporin C with DAOC/DACS were carried out under <sup>18</sup>O<sub>2</sub> or in H<sub>2</sub><sup>18</sup>O. Incorporation of <sup>18</sup>O-label into the products [3-<sup>13</sup>C]DAC, [3-<sup>13</sup>C,4-²H]-3β-hydroxycepham and 3β-spiroepoxide cepham was observed from both sources. The results suggest that intermediates capable of oxygen-exchange are formed during the enzymatic reactions.</p> <p>Two substrate analogues, the 5-epipenicillin N and the 2β-difluoromethyl penicillin N, have been synthesised in order to probe the substrate specificity of DAOC/DACS with respect to the ring-expansion activity. The 5-epipenicillin N was not accepted as a substrate by DAOC/DACS, and the observations made indicate that it was unstable under the incubation conditions. No product was either observed from incubations of the 2β-difluoromethyl penicillin N with DAOC/DACS, although bioassay tests suggested a cephem product had been formed in very small amounts.</p> <p>Finally, the results of a substrate specificity comparison between the soluble recombinant enzymes deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS) from <em>Cephalosporium acremonium</em> and deacetoxycephalosporin C synthase (DAOCS) from <em>Streptomyces clavuligerus</em> are described.</p>
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