Characterization of five catalytic activities associated with the NADPH : 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxyl ase of the Xanthobacter strain Py2 epoxide carboxylase

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropa ne followed by carboxylation to acetoacetate. Epoxypropane carboxylation is a minimetabolic pathway that requires four enzymes, NADPH, NAD(+), and coe nzyme M (CoM; 2-mercaptoethanesulfonate) and occurs with the overall reacti on stoichiometry: epoxypropane + CO2 + NADPH + NAD(+) + CoM --> acetoacetat e + H+ + NADP(+) + NADH + CoM. The terminal enzyme of the pathway is NADPH: 2-ketopropyl-CoM [2-(2-keropropylthio)ethanesulfonate] oxidoreductase/carbo xylase (2-KPCC), an FAD-containing enzyme that is a member of the NADPH:dis ulfide oxidoreductase family of enzymes and that catalyzes the reductive cl eavage and carboxylation of 2-ketopropyl-CoM to form acetoacetate and CoM a ccording to the reaction: 2-ketopropyl-CoM + NADPH + CO2 --> acetoacetate NADP(+) + CoM. In the present work, 2-KPCC has been characterized with res pect to the above reaction and four newly discovered partial reactions of r elevance to the catalytic mechanism, and each of which requires the formati on of a stabilized enolacetone intermediate. These four reactions are (1) N ADPH-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-ketop ropyl-CoM from CoM and acetoacetate, the reverse of the physiologically imp ortant forward reaction; (3) acetoacetate decarboxylation to form acetone a nd CO2; and (4) acetoacetate/(CO2)-C-14 exchange to form C-14(1)-acetoaceta te and CO2. Acetoacetate decarboxylation and (CO2)-C-14 exchange occurred i ndependent of NADP(H) and CoM, demonstrating that these substrates are not central to the mechanism of enolate generation and stabilization. 2-KPCC di d nor uncouple NADPH oxidation or NADP(+) reduction from the reactions invo lving cleavage or formation of 2-ketopropyl-CoM. N-Ethylmaleimide inactivat ed the reactions forming/using 2-ketopropyl-CoM but did not inactivate acet oacetate decarboxylation or (CO2)-C-14 exchange reactions. The biochemical characterization of 2-KPCC and the associated five catalytic activities has allowed the formulation of an unprecedented mechanism of substrate activat ion and carboxylation that involves NADPH oxidation, a redox active disulfi de, thiol-mediated reductive cleavage of a C-S thioether bond, the formatio n of a CoM:cysteine mixed disulfide, and enolacetone stabilization.