Purification and characterization of 1-naphthol-2-hydroxylase from carbaryl-degrading Pseudomonas strain C4
DSpace at IIT Bombay
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Title |
Purification and characterization of 1-naphthol-2-hydroxylase from carbaryl-degrading Pseudomonas strain C4
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Creator |
SWETHA, VP
BASU, A PHALE, PS |
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Subject |
p-hydroxybenzoate hydroxylase
fad-dependent monooxygenase oxidative half-reaction phenol hydroxylase bacterial metabolism mechanism 1-naphthol oxygenase component degradation |
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Description |
Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274,375, and 445 run, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants K-m and V-max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 mu M and 9.5 and 5.1 mu mol min(-1) mg(-1), respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The K-i for 1-naphthol was determined to be 79.8 mu M. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
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Publisher |
AMER SOC MICROBIOLOGY
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Date |
2011-07-17T06:13:16Z
2011-12-26T12:50:09Z 2011-12-27T05:36:11Z 2011-07-17T06:13:16Z 2011-12-26T12:50:09Z 2011-12-27T05:36:11Z 2007 |
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Type |
Article
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Identifier |
JOURNAL OF BACTERIOLOGY, 189(7), 2660-2666
0021-9193 http://dx.doi.org/10.1128/JB.01418-06 http://dspace.library.iitb.ac.in/xmlui/handle/10054/4637 http://hdl.handle.net/10054/4637 |
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Language |
en
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