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 K(m) values for maleylacetate and NADH were 58 and 30 muM, 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 V(max) value, but its K(m) value was estimated to be 77 muM. 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.