title: 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.
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218.
authors.forename: S P
authors.initials: SP
authors.lastname: Salowe
authors.forename: W J
authors.initials: WJ
authors.lastname: Krol
authors.forename: D
authors.initials: D
authors.lastname: Iwata-Reuyl
authors.forename: C A
authors.initials: CA
authors.lastname: Townsend
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D002250:Carbon Radioisotopes; D002969:Clavulanic Acids / Q000096:biosynthesis* / Q000138:chemical synthesis; D007202:Indicators and Reagents; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011865:Radioisotope Dilution Technique; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology; D014316:Tritium
chemical_list: D000900:Anti-Bacterial Agents; D002250:Carbon Radioisotopes; D002969:Clavulanic Acids; D007202:Indicators and Reagents; D014316:Tritium; D006899:Mixed Function Oxygenases; C066046:clavaminate synthase