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1-Naphthol 2-hydroxylase from Pseudomonas sp. strain C6: purification, characterization and chemical modification studies

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Abstract

1-Naphthol 2-hydroxylase (1-NH) which catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene was purified to homogeneity from carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a homodimer with subunit molecular weight of 66 kDa. UV, visible and fluorescence spectral properties, identification of flavin moiety by HPLC as FAD, and reconstitution of apoenzyme by FAD suggest that enzyme is FAD-dependent. 1-NH accepts electron from NADH as well as NADPH. Besides 1-naphthol (K m, 9.1 μM), the enzyme also accepts 5-amino 1-naphthol (K m, 6.4 μM) and 4-chloro 1-naphthol (K m, 2.3 μM) as substrates. Enzyme showed substrate inhibition phenomenon at high concentration of 1-naphthol (K i, 283 μM). Stoichiometric consumption of oxygen and NADH, and biochemical properties suggest that 1-NH belongs to FAD containing external flavomonooxygenase group of oxido-reductase class of enzymes. Based on biochemical and kinetic properties, 1-NH from Pseudomonas sp. strain C6 appears to be different than that reported earlier from Pseudomonas sp. strain C4. Chemical modification and protection by 1-naphthol and NADH suggest that His, Arg, Cys, Tyr and Trp are at or near the active site of 1-NH.

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Acknowledgments

We thank Profs. Bhat PJ, Punekar NS, Mr. Rahul Srivastav and Randhir Singh, IIT-B for valuable discussion. Technical grade Carbaryl was a gift from Bayer (India) Ltd.

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  1. Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, 400 076, India

    Shivjee Sah & Prashant S. Phale

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  1. Shivjee Sah
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  2. Prashant S. Phale
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Correspondence to Prashant S. Phale.

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Sah, S., Phale, P.S. 1-Naphthol 2-hydroxylase from Pseudomonas sp. strain C6: purification, characterization and chemical modification studies. Biodegradation 22, 517–526 (2011). https://doi.org/10.1007/s10532-010-9424-2

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  • DOI: https://doi.org/10.1007/s10532-010-9424-2

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