Degradation of chloroaromatics: purification and characterization of maleylacetate reductase from Pseudomonas sp. strain B13.
Maleylacetate reductase of Pseudomonas sp. strain B13 was purified to homogeneity by chromatography on DEAE-cellulose, Butyl-Sepharose, Blue-Sepharose, and Sephacryl S100. The final preparation gave a single band by polyacrylamide gel electrophoresis under denaturing conditions and a single symmetrical peak by gel filtration under nondenaturing conditions. The subunit M(r) value was 37,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Estimation of the native M(r) value by gel filtration gave a value of 74,000 with a Superose 6 column, indicating that the enzyme is dimeric. The pH and temperature optima were 5.4 and 50 degrees C, respectively. The pI of the enzyme was estimated to be 7.0. The apparent Km values for maleylacetate and NADH were 58 and 30 microM, respectively, and the maximum velocity was 832 U/mg of protein for maleylacetate. Maleylacetate and various substituted maleylacetates, such as 2-chloro- and 2-methyl-maleylacetate, were reduced at significant rates. NADPH was also used as a cofactor instead of NADH with nearly the same Vmax value, but its Km value was estimated to be 77 microM. Reductase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group in the enzyme.
Kaschabek
S R
SR
Chemische Mikrobiologie der Bergischen Universität--Gesamthochschule Wuppertal, Germany.
Reineke
W
W
D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics
D006843:Hydrocarbons, Chlorinated; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C021530:maleylacetate reductase