Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia.
2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.
Radjendirane
V
V
Department of Biochemistry, Indian Institute of Science, Bangalore.
Bhat
M A
MA
Vaidyanathan
C S
CS
D001051:Apoenzymes / Q000302:isolation & purification; D002846:Chromatography, Affinity; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology
D001051:Apoenzymes; D008670:Metals; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D014227:Triazines; C003213:Procion Brilliant Red M-2BS; D009249:NADP; C012842:Cibacron Blue F 3GA; D006899:Mixed Function Oxygenases; C034354:2,4-dichlorophenol hydroxylase