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Natural α,β-epoxyketone proteasome inhibitors produced primarily by Streptomyces species, such as TMC-86A, are potent anticancer compounds. The enzymes responsible for biosynthesis of their key α,β-epoxyketone pharmacophore had yet to be determined at the onset of this research. Elucidation of the responsible enzymes and their putative substrate would allow for stereoselective completion of a synthetically challenging epoxidation reaction, responsible for the high cost of epoxyketone pharmaceuticals produced by industry. The cytochrome P450 TmcI and the flavin-dependent TmcF enzymes from the TMC- 86A biosynthetic gene cluster in Streptomyces chromofuscus, believed to have a role in epoxyketone biosynthesis, were cloned, expressed and overproduced. Potential substrates were produced through a peptide coupling synthetic route. In vitro assays demonstrated that EpnF, a TmcF homologue, could produce α,β-epoxyketone compounds with an α-dimethyl-β-keto carboxylic acid substrate. These substrates were prone to decarboxylative degradation, therefore stable methyl ester precursors were synthesised as an alternative. Using an esterase, methyl ester precursors were hydrolysed to reproduce the previously observed epoxyketone compound in a one pot reaction with EpnF. Substrate specificity of EpnF was probed by in vitro assay using an alanine derived analogue, which was found to be a viable substrate for the enzyme. Lastly, the EpnF catalytic cycle was interrogated with a synthesised authentic standard of a proposed intermediate, which was observed to co-elute with the desired intermediate from a quenched EpnF assay using UHPLC-MS chromatography.