Elucidation of the order of oxidations and identification of an intermediate in the multistep clavaminate synthase reaction

Abstract

The enzyme clavaminate synthase (CS) catalyzes the formation of the first bicyclic intermediate in the biosynthetic pathway to the potent beta-lactamase inhibitor clavulanic acid. Our previous work has led to the proposal that the cyclization/desaturation of the substrate proclavaminate proceeds in two oxidative steps, each coupled to a decarboxylation of alpha-ketoglutarate and a reduction of dioxygen to water [Salowe, S. P., Marsh, E. N., & Townsend, C. A. (1990) Biochemistry 29, 6499-6508]. We have now employed kinetic isotope effect studies to determine the order of oxidations for CS purified from Streptomyces clavuligerus. By using (4'RS)-[4'-3H,1-14C]-rac-proclavaminate, a primary T(V/K) = 8.3 +/- 0.2 was measured from [3H]water release data, while an alpha-secondary T(V/K) = 1.06 +/- 0.01 was determined from the changing 3H/14C ratio of the product clavaminate. Values for the primary and alpha-secondary effects of 11.9 +/- 1.7 and 1.12 +/- 0.07, respectively, were obtained from the changing 3H/14C ratio of the residual proclavaminate by using new equations derived for a racemic substrate bearing isotopic label at both primary and alpha-secondary positions. Since only the first step of consecutive irreversible reactions will exhibit a V/K isotope effect, we conclude that C-4' is the initial site of oxidation in proclavaminate. As expected, no significant changes in the 3H/14C ratio of residual substrate were observed with [3-3H,1-14C]-rac-proclavaminate. However, two new tritiated compounds were produced in this incubation, apparently the result of isotope-induced branching brought about by the presence of tritium at the site of the second oxidation. One of these compounds was identified by comparison to authentic material as dihydroclavaminate, a stable intermediate that normally remains enzyme-bound. On the basis of the body of information available and the similarities to alpha-ketoglutarate-dependent dioxygenases, a comprehensive mechanistic scheme for CS is proposed to account for this unusual enzymatic transformation.

Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis

“…Protected deoxyproclavaminic acid (compound 9) was prepared as shown in Fig. 3 from the known ornithine derivative compound 7 (18) by our reported procedure for ␤-lactam formation (31). The ␤-lactam compound 9 (345 mg, 0.841 mmol) was dissolved in 50 ml of 1:1 ethanol-H 2 O, and 85 mg of 10% Pd/C (Aldrich Chemical Co., Milwaukee, Wis.) was added.…”

Biosynthesis of clavam metabolites

“…The final two early steps leading to clavaminic acid formation are both catalyzed by CAS. First, proclavaminic acid undergoes cyclization to produce dihydroclavaminic acid, which is then desaturated to produce clavaminic acid [61].…”

Five Additional Genes Are Involved in Clavulanic Acid Biosynthesis in Streptomyces clavuligerus

“…Genes located in this region include ceaS (encoding carboxyethylarginine synthase) (14), bls (␤-lactam synthetase) (3,18), pah (proclavaminate amidinohydrolase) (2,11,34), cas2 (clavaminate synthase) (4,17,26,27), and cad (clavulanic acid dehydrogenase) (21), all enzymes for recognized steps in the biosynthetic pathway. In addition, oat, a gene encoding a protein with ornithine acetyltransferase activity, has been identified (13), but its role in clavulanic acid is not yet understood, and claR, a gene encoding a transcriptional regulator that controls the late steps in clavulanic acid biosynthesis, is also located in this region (22,24).…”