Purification and Characterization of Clavaminate Synthase from Streptomyces antibioticus

Abstract

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.

Enzymes Catalyzing the Early Steps of Clavulanic Acid Biosynthesis Are Encoded by Two Sets of Paralogous Genes in Streptomyces clavuligerus

“…2), and evidence has been obtained to indicate that both clavulanic acid and the antipodal clavam metabolites share a common pathway, at least to the level of proclavaminate or clavaminate (5,9). The structural similarity between clavulanic acid and the other clavam metabolites, together with the evidence for a shared pathway, has led to the assumption that these compounds comprise a family of biosynthetically related metabolites.…”

Measurement of the Degree of Coupled Isotopic Enrichment of Different Positions in an Antibiotic Peptide by NMR

“…C14 has an calculate F ( C,N ) (times x) for C3 (and C10), using Eq. [3]. effective J CN of 8 Hz but is discussed further below, as it xF ( C,N ) was 0.10 for C10 and F ( C,N ) was 0.12 for C3 (Table is coupled Fig.…”

“…The function of CT, and fitting the results to Eq. [3]. experiments are also relatively sensitive because they emIn principle, this procedure yields estimates of both the ploy 1 H excitation and detection, while nonetheless permit-magnitude of J CN and x times the fraction of 13 C bound to ting resolution of signals in 13 C (and to measure x and F ( C,N ) .…”

“…HMQC has been used to distinguish between small mole-NMR spectroscopy can provide information as to what cules with and without 15 N at a given position by allowing sites have been labeled with NMR active isotopes, and to 13 C to evolve under coupling to 15 N for a period of 1/2J, what degree (3). NMR can also reveal the fraction of mole-with the result that the signal intensity is either 1 or 0 decules in which a labeled site is close to another labeled pending on whether the 13 C is coupled to 15 N (4).…”

“…The fits to Eq. [3] were performed using KaleidaGraph 2.0 and yielded J CN Å 12.4 and 5.6 Hz, respectively. The proton chemical shifts quoted here are 0.14 ppm ( ÅJ HC /2) from the values listed in Table 1, because the spectra used here were not decoupled with respect to 13 C.…”