Probable Role of Clavaminic Acid as the Terminal Intermediate in the Common Pathway to Clavulanic Acid and the Antipodal Clavam Metabolites

Emerging chemical and genetic evidence suggests that separate biochemical solutions have evolved to synthesize the four known classes of β-lactam antibiotics. One of these classes contains clavulanic acid ( 1 ) and a family of structurally related but antipodal clavam metabolites 2 − 5, 7, and 8 which lack a carboxylate at C-3, have a different oxidation state, and exhibit stereochemical features at C-2. Previous work has demonstrated the incorporation of ornithine/arginine in the identical regiochemical sense in all of these natural products, and has established the common intermediacy of the monocyclic β-lactam proclavaminic acid ( 12 ) as well. In this paper the quite advanced bicyclic intermediate clavaminic acid ( 14 ) has been synthesized in doubly 13 C-labeled form by preparative incubation of recombinant clavaminate synthase. The intact and equally efficient incorporation of 14 into valclavam ( 7 ) and 2-(2-hydroxyethyl)clavam ( 8 ), together with 18 O 2 -incorporation experiments, has been interpreted to define clavaminic acid as the final intermediate shared in the biosynthesis of clavulanic acid and the antipodal clavams. A mechanistic rationale of this interrelationship and the late stages of the respective biosyntheses is proposed.