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Journal of Bacteriology, April 2007, p. 2660-2666, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01418-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4{triangledown}

Vandana P. Swetha, Aditya Basu, and Prashant S. Phale*

Biotechnology Group, School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India

Received 7 September 2006/ Accepted 9 January 2007

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 nm, 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 Km and Vmax for 1-naphthol and NADPH were determined to be 9.6 and 34.2 µM and 9.5 and 5.1 µmol min–1 mg–1, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-naphthol was determined to be 79.8 µM. 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.

* Corresponding author. Mailing address: Biotechnology Group, School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India. Phone: 91-22-2576 7836. Fax: 91-22-2572 3480. E-mail: pphale{at}iitb.ac.in.

{triangledown} Published ahead of print on 19 January 2007.

Journal of Bacteriology, April 2007, p. 2660-2666, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01418-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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