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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 13C-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 18O2-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.

Clavaminate synthase (CS), a key enzyme in the clavulanic acid biosynthetic pathway, has been purified to electrophoretic homogeneity from Streptomyces antibioticus (Tü 1718), a species that does not produce clavulanic acid. A comparison of the physical and kinetic properties of clavaminate synthase from S. antibioticus (CS3) and the two isozymes from Streptomyces clavuligerus (CS1 and CS2) has been conducted. In oxidative reactions requiring the co-substrates O2, alpha-ketoglutaric acid, and catalytic Fe2+, both CS1 and CS2 catalyze three distinct transformations, the hydroxylation of deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and the cyclization and desaturation of proclavaminic acid to clavaminic acid. We have demonstrated that CS3 from S. antibioticus also catalyzes these three oxidations. The apparent molecular mass of CS3 from matrix-assisted laser desorption mass spectrometry is 35,839 +/- 36 Da. The enzyme is a monomer in solution as determined by gel filtration chromatography. Analysis of the four possible proclavaminic acid diastereomers confirmed the absolute configuration of the substrate to be 2S,3R. Based upon N-terminal sequence comparisons among the three proteins, CS3 possesses the higher degree of homology with the CS1 isozyme from S. clavuligerus. Although previously associated solely with clavulanic acid biosynthesis, we propose these findings and recent precursor incorporation data support the view that clavaminate synthase plays a critical role in the biosynthesis of the clavam metabolites.

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