Purification and characterization of maleylacetate reductase from Alcaligenes eutrophus JMP134(pJP4).
Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enzyme was purified to apparent homogeneity from an extract of 2,4-dichlorophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a dimer of two identical subunits of 35 kDa. The pI was determined to be at pH 5.4. There was no indication of a flavin prosthetic group. The enzyme was inactivated by p-chloromercuribenzoate but not by EDTA, 1,10-phenanthroline, or dithiothreitol. Maleylacetate and 2-chloromaleylacetate were converted with similar efficiencies (with NADH as cosubstrate, Km = 31 microM for each substrate and kcat = 8,785 and 7,280/min, respectively). NADH was preferred to NADPH as the cosubstrate. Upon reduction of 2-chloramaleylacetate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NADH. These results and the kinetic parameters suggest that the maleylacetate reductase is sufficient to channel the 2,4-D degradation intermediate 2-chloromaleylacetate into the 3-oxoadipate pathway. In a data base search the NH2-terminal sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in 2,4-D degradation.
Seibert
V
V
Institut für Mikrobiologie, Universität Stuttgart, Germany.
Stadler-Fritzsche
K
K
Schlömann
M
M
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004589:Electrophoresis, Disc; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014462:Ultrafiltration
D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C021530:maleylacetate reductase