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The purification and characterization of 4-ethylphenol methylenehydroxylase, a flavocytochrome from Pseudomonas
C D Reeve1, M A Carver, D J Hopper
1Department of Biochemistry, University College of Wales, U.K.
Abstract:
The enzyme 4-ethylphenol methylenehydroxylase was purified from Pseudomonas putida JD1 grown on 4-ethylphenol. It is a flavocytochrome c for which the Mr was found to be 120,000 by ultracentrifuging and 126,000 by gel filtration. The enzyme consists of two flavoprotein subunits each of Mr 50,000 and two cytochrome c subunits each of Mr 10,000. The redox potential of the cytochrome is 240 mV. Hydroxylation proceeds by dehydrogenation and hydration to give 1-(4'-hydroxyphenyl)ethanol, which is also dehydrogenated by the same enzyme to 4-hydroxyacetophenone. The enzyme will hydroxylate p-cresol but is more active with alkylphenols with longer-chain alkyl groups. It is located in the periplasm of the bacterium.
Insights
Researchers purified 4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1. This flavocytochrome c enzyme hydroxylates alkylphenols, converting them into valuable compounds.
Area of Science:
- Biochemistry
- Enzymology
- Microbial Metabolism
Background:
- Pseudomonas putida JD1 metabolizes alkylphenols.
- Enzymes involved in alkylphenol degradation are crucial for bioremediation and industrial applications.
Purpose of the Study:
- To purify and characterize the enzyme responsible for 4-ethylphenol hydroxylation from Pseudomonas putida JD1.
- To elucidate the enzyme's structure, properties, and catalytic mechanism.
Main Methods:
- Enzyme purification using ultracentrifugation and gel filtration.
- Characterization of enzyme subunits and redox potential.
- Analysis of hydroxylation reaction products.
Main Results:
- Purification of 4-ethylphenol methylenehydroxylase, a flavocytochrome c (Mr 120,000-126,000).
- The enzyme comprises two flavoprotein subunits (Mr 50,000) and two cytochrome c subunits (Mr 10,000).
- The enzyme catalyzes the conversion of 4-ethylphenol to 1-(4'-hydroxyphenyl)ethanol and subsequently to 4-hydroxyacetophenone, and also hydroxylates p-cresol.
Conclusions:
- 4-ethylphenol methylenehydroxylase is a periplasmic enzyme with a complex subunit structure.
- The enzyme exhibits broad substrate specificity for alkylphenols, with higher activity for longer alkyl chains.
- This enzyme represents a key component in the microbial degradation pathway of alkylphenols.