Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

Radjendirane, V and Bhat, Manzoor A and Vaidyanathan, CS (1991) Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia. In: Archives of Biochemistry and Biophysics, 288 (1). pp. 169-176.

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Abstract

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-dicholorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 μImage for 2,4-dichlorophenol, and 100 μImage 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.

Item Type:	Journal Article
Publication:	Archives of Biochemistry and Biophysics
Publisher:	Elsevier science
Additional Information:	Copyright of this article belongs to Elsevier science.
Keywords:	Para-Hydroxybenzoate Hydroxylase;Phenol Hydroxylase; 3-Hydroxybenzoate 6-Hydroxylase;Salicylate Hydroxylase; Enzyme;Flavoprotein;Fluorescens;Aeruginosa;Blue.
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	10 Nov 2010 06:11
Last Modified:	10 Nov 2010 06:11
URI:	http://eprints.iisc.ac.in/id/eprint/33597

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