Purification to homogeneity and reconstitution of the individual components of the epoxide carboxylase multiprotein enzyme complex from Xanthobacter strain Py2.
Epoxide metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by an NADPH- and NAD+-dependent carboxylation reaction that forms beta-keto acids as products. Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity. One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase. In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized. These component proteins are as follows: component I, a homohexameric protein consisting of 41.7-kDa subunits; component III, a dimeric protein consisting of 26.0- and 26.2-kDa polypeptides; and component IV, a dimeric protein consisting of a single 25.4-kDa polypeptide. Component I contained 5 mol of tightly bound zinc per mol of protein. Component I was specifically inactivated by methylepoxypropane, a time-dependent irreversible inactivator of epoxide carboxylase activity, suggesting that this component plays an integral role in epoxide binding and activation. No metals or organic cofactors were detected for components III and IV. The molecular weights, N-terminal sequences, and amino acid compositions of the purified epoxide carboxylase components were determined and found to correlate with open reading frames within and adjacent to a cloned fragment of DNA that complements Xanthobacter Py2 mutants defective in epoxide degradation. Using the purified epoxide carboxylase system, epoxide carboxylation was found to be stoichiometrically coupled to the transhydrogenation of pyridine nucleotide cofactors according to the following equation: epoxypropane + CO2 + NADPH + NAD+ --> acetoacetate + H+ + NADP+ + NADH.
Allen
J R
JR
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Ensign
S A
SA
D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D016366:Open Reading Frames
D004269:DNA, Bacterial; D002262:Carboxy-Lyases; C109739:epoxide carboxylase