title: Characterization of five catalytic activities associated with the NADPH:2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase of the Xanthobacter strain Py2 epoxide carboxylase system.
abstract: The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by carboxylation to acetoacetate. Epoxypropane carboxylation is a minimetabolic pathway that requires four enzymes, NADPH, NAD(+), and coenzyme M (CoM; 2-mercaptoethanesulfonate) and occurs with the overall reaction stoichiometry: epoxypropane + CO(2) + NADPH + NAD(+) + CoM --> acetoacetate + H(+) + NADP(+) + NADH + CoM. The terminal enzyme of the pathway is NADPH:2-ketopropyl-CoM [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase (2-KPCC), an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase family of enzymes and that catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-CoM to form acetoacetate and CoM according to the reaction: 2-ketopropyl-CoM + NADPH + CO(2) --> acetoacetate + NADP(+) + CoM. In the present work, 2-KPCC has been characterized with respect to the above reaction and four newly discovered partial reactions of relevance to the catalytic mechanism, and each of which requires the formation of a stabilized enolacetone intermediate. These four reactions are (1) NADPH-dependent cleavage and protonation of 2-ketopropyl-CoM to form NADP(+), CoM, and acetone, a reaction analogous to the physiological reaction but in which H(+) is the electrophile; (2) NADP(+)-dependent synthesis of 2-ketopropyl-CoM from CoM and acetoacetate, the reverse of the physiologically important forward reaction; (3) acetoacetate decarboxylation to form acetone and CO(2); and (4) acetoacetate/(14)CO(2) exchange to form (14)C(1)-acetoacetate and CO(2). Acetoacetate decarboxylation and (14)CO(2) exchange occurred independent of NADP(H) and CoM, demonstrating that these substrates are not central to the mechanism of enolate generation and stabilization. 2-KPCC did not uncouple NADPH oxidation or NADP(+) reduction from the reactions involving cleavage or formation of 2-ketopropyl-CoM. N-Ethylmaleimide inactivated the reactions forming/using 2-ketopropyl-CoM but did not inactivate acetoacetate decarboxylation or (14)CO(2) exchange reactions. The biochemical characterization of 2-KPCC and the associated five catalytic activities has allowed the formulation of an unprecedented mechanism of substrate activation and carboxylation that involves NADPH oxidation, a redox active disulfide, thiol-mediated reductive cleavage of a C-S thioether bond, the formation of a CoM:cysteine mixed disulfide, and enolacetone stabilization.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
authors.forename: D D
authors.initials: DD
authors.lastname: Clark
authors.forename: J R
authors.initials: JR
authors.lastname: Allen
authors.forename: S A
authors.initials: SA
authors.lastname: Ensign
mesh_terms: D000090:Acetoacetates / Q000737:chemistry / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D002250:Carbon Radioisotopes; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020536:Enzyme Activators / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D015080:Mesna / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry; D013379:Substrate Specificity; D020584:Xanthobacter / Q000201:enzymology*
chemical_list: D000090:Acetoacetates; D002250:Carbon Radioisotopes; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D011522:Protons; D012545:Schiff Bases; D002245:Carbon Dioxide; C016635:acetoacetic acid; D009249:NADP; D010088:Oxidoreductases; D002262:Carboxy-Lyases; C109739:epoxide carboxylase; D019746:Intramolecular Oxidoreductases; C102324:epoxypropane isomerase; D015080:Mesna; D005033:Ethylmaleimide