A Flavin-Dependent Decarboxylase–Dehydrogenase–Monooxygenase Assembles the Warhead of α,β-Epoxyketone Proteasome Inhibitors

Abstract

The α,β-epoxyketone proteasome inhibitor TMC-86A was discovered as a previously unreported metabolite of Streptomyces chromofuscus ATCC49982, and the gene cluster responsible for its biosynthesis was identified via genome sequencing. Incorporation experiments with [(13)C-methyl]l-methionine implicated an α-dimethyl-β-keto acid intermediate in the biosynthesis of TMC-86A. Incubation of the chemically synthesized α-dimethyl-β-keto acid with a purified recombinant flavin-dependent enzyme that is conserved in all known pathways for epoxyketone biosynthesis resulted in formation of the corresponding α-methyl-α,β-epoxyketone. This transformation appears to proceed via an unprecedented decarboxylation-dehydrogenation-monooxygenation cascade. The biosynthesis of the TMC-86A warhead is completed by cytochrome P450-mediated hydroxylation of the α-methyl-α,β-epoxyketone.

Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism

“…These include simple oxidations of thiols by oxidases to form thiocarbonyls [35], thereby extending the chemistry of oxidases beyond oxidation of C-O, C-C and C-N bonds. Furthermore, complex cascade chemistry has been postulated to occur in a flavin dependent decarboxylase-dehydrogenase-monooxygenase during assembly of the warhead a,b-epoxyketone proteasome inhibitors [36] and for the multifunctional monooxygenase XanO4 that catalyzes sequential insertion of two oxygen atoms and a cryptic demethoxylation in the biosynthesis of xantholipids [37]. These intriguing reactions extend further the already large family of biocatalysts represented by the flavin-dependent monooxygenases [22].…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…270,271 An example of this chemistry is found in the biosynthesis of the a,b-epoxyketone proteasome inhibitor TMC-86A (a mixed NRPS/PKS pathway), in which the P450 enzyme TmcI performs the hydroxylation of the methyl group adjacent to the epoxy moiety in this compound. 239 The biosynthesis of the related compound eponemycin also contains a homologous P450 that is believed to perform the analogous reaction; such a P450 is absent in the epoxomicin gene cluster that lacks this hydroxyl group. 3.6.2.2.2 Introduction of double bonds -WS9326A biosynthesis.…”

“…239 In this case, evidence suggests that P450 TmcK introduces a double bond into the side chain of Leu-2 aer the amino acid is PCP-bound, with further experiments no doubt required to probe this unusual PCP-bound transformation. 3.6.2.1.2 Phenolic coupling of PCP-bound peptides.…”

The Tripod for Bacterial Natural Product Discovery: Genome Mining, Silent Pathway Induction, and Mass Spectrometry-Based Molecular Networking

“…This is prompting the discovery of new biosynthetic and regulatory strategies in these microorganisms (8). Still, many biosynthetic enzymes are unprecedented for their catalytic activities and display only distant homologues or orthologues with known three-dimensional structures and validated catalytic mechanisms (as an example, please see reference 10). Even in well-known secondary metabolite producers, the organization of BGCs coding for secondary metabolites is being reviewed, and unexpected biosynthetic setups are being found (11).…”