Identification and characterization of epoxide carboxylase activity in cell extracts of Nocardia corallina B276

Abstract

The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete Nocardia corallina B276 was investigated. Suspensions of N. corallina cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of N. corallina was stimulated by the addition of CO2 and inhibited by the depletion of CO2. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO2, NAD+, and a reductant (NADPH or dithiothreitol). In the absence of CO2, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in N. corallina occurs by a carboxylation reaction forming beta-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium Xanthobacter strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121-32128, 1997). The addition of epoxide carboxylase component I from Xanthobacter strain Py2 to methylepoxypropane-inactivated N. corallina extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II from Xanthobacter Py2 to active N. corallina extracts stimulated epoxide isomerase rates to the same levels observed with the purified Xanthobacter system. Antibodies raised against Xanthobacter strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grown N. corallina extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO2 fixation and the activity of a multicomponent epoxide carboxylase enzyme system.

FIG. 1

Requirement of CO₂ for epoxyalkane degradation by propylene-grown N. corallina. Assays were performed with whole-cell suspensions (0.5 mg of protein). Closed symbols, assays performed with CO₂ and NaHCO₃ (50 mM combined concentration); open symbols, assays performed without CO₂ and NaHCO₃; squares, epoxypropane remaining; circles, 1,2-epoxybutane remaining.

FIG. 2

Effect of CO₂ on the time courses of epoxypropane degradation and acetoacetate formation catalyzed by cell extracts of N. corallina. Assays were performed with 7.5 mg of cell extract. Closed symbols, assays performed with CO₂ and NaHCO₃ (60 mM); open symbols, assays performed without CO₂ and NaHCO₃; squares, epoxypropane remaining; circles, acetoacetate formed; triangles, acetone formed.

FIG. 3

Requirement of new protein synthesis for the degradation of propylene and epoxypropane by N. corallina grown with glucose as the carbon source. Assays were conducted in 150-ml serum bottles containing 15 ml of glucose-grown cells (A₆₀₀ = 1.47) or propylene-grown cells (A₆₀₀ = 0.93) that had been transferred from 200-ml cultures grown in shake flasks. Closed symbols, assay bottles containing chloramphenicol (10 mg) and rifampin (5 mg); open symbols, assay bottles without chloramphenicol or rifampin present; circles, propylene-grown cells; squares, glucose-grown cells. (A) Propylene remaining; (B) epoxypropane remaining.

FIG. 4

Gel electrophoretic analysis of propylene-induced polypeptides in N. corallina and comparison to purified epoxide carboxylase components (comp.) I and II from Xanthobacter strain Py2. Lane 1, molecular mass standards (2 μg each); lane 2, epoxide carboxylase component I from Xanthobacter strain Py2 (3 μg); lane 3, epoxide carboxylase component II from Xanthobacter strain Py2 (3 μg); lane 4, glucose-grown cell extract from Xanthobacter strain Py2 (25 μg); lane 5, propylene-grown cell extract from Xanthobacter strain Py2 (25 μg); lane 6, glucose-grown cell extract from N. corallina (25 μg); lane 7, propylene-grown cell extract from N. corallina (25 μg); lane 8, epoxide carboxylase components I (3 μg) and II (3 μg) from Xanthobacter strain Py2.

FIG. 5

Immunoblot analysis of glucose- and propylene-grown cell extracts of N. corallina. (A) Immunoblot prepared using antibodies raised against epoxide carboxylase component I from Xanthobacter strain Py2 as the probe. The arrow indicates the position of component I. Lanes 1 and 6, epoxide carboxylase component I from Xanthobacter strain Py2 (0.5 μg); lane 2, glucose-grown cell extract from Xanthobacter strain Py2 (15 μg); lane 3, propylene-grown cell extract from Xanthobacter strain Py2 (15 μg); lane 4, glucose-grown cell extract from N. corallina (15 μg); lane 5, propylene-grown cell extract from N. corallina (15 μg). (B) Immunoblot prepared using antibodies raised against epoxide carboxylase component II from Xanthobacter strain Py2 as the probe. The arrow indicates the position of component II. Lane 1, glucose-grown cell extract from N. corallina (15 μg); lane 2, propylene-grown cell extract from N. corallina (15 μg); lane 3, epoxide carboxylase component II from Xanthobacter strain Py2 (0.5 μg).

FIG. 6

Concentration- and time-dependent inactivation of epoxide carboxylase in whole-cell suspensions of N. corallina by methylepoxypropane. Each assay mixture contained cell suspension (0.35 mg of protein) and CO₂ and NaHCO₃ (50 mM total). Assays were initiated by the addition of 2 μmol of epoxypropane. Symbols: ▪, no methylepoxypropane; ○, 0.5 mM methylepoxypropane; ▴, 1 mM methylepoxypropane; ◊, 2 mM methylepoxypropane; ▾, 4 mM methylepoxypropane.

FIG. 7

Restoration of epoxide carboxylase activity in methylepoxypropane-treated cell extracts of N. corallina by addition of epoxide carboxylase component I from Xanthobacter strain Py2. Symbols: ▪, assays performed with CO₂ and NaHCO₃ (60 mM); □, assays performed without CO₂ and NaHCO₃. (A) Assays of cell extract (4.8 mg) prepared from methylepoxypropane-treated N. corallina; (B) assays of cell extract (5.4 mg) prepared from methylepoxypropane-treated Xanthobacter strain Py2.

FIG. 8

Epoxide carboxylase component II from Xanthobacter strain Py2 confers epoxide isomerase activity in cell extracts of N. corallina. Assays were performed with cell extracts of propylene-grown N. corallina (7.5 mg) in the absence of CO₂. Closed symbols, epoxypropane remaining; open symbols, acetone produced; inverted triangles, addition of epoxide carboxylase component I (0.8 mg); squares, addition of epoxide carboxylase component II (0.6 mg); circles, addition of epoxide carboxylase component III (0.5 mg); triangles, addition of epoxide carboxylase component IV (0.6 mg).