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Kinetic and Spectroscopic Characterization of 1-Naphthol 2-hydroxylase from Pseudomonas sp. Strain C5

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Abstract

1-Naphthol 2-hydroxylase (1-NH) catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene. 1-NH from carbaryl degrading Pseudomonas strain C5 was purified and characterized for its kinetic and spectroscopic properties. The enzyme was found to be NAD(P)H-dependent external flavin monooxygenase. Though the kinetic parameters of 1-NH from strain C5 appear to be similar to 1-NH enzyme from strains C4 and C6, however, they differ in their N-terminal sequences, mole content of flavin adenine dinucleotide (FAD), reconstitution of apoenzyme, and K i. 1-NH showed narrow substrate specificity with comparable hydroxylation efficiency on 1-naphthol and 5-amino 1-naphthol (~30 %) followed by 4-chloro 1-naphthol (~9 %). Salicylate was found to be the nonsubstrate effector. The flavin fluorescence of 1-NH was found to increase in the presence of 1-naphthol (K d = 11.3 μM) and salicylate (K d = 1027 μM). The circular dichroism (CD) spectra showed significant perturbations in the presence of NAD(P)H, whereas no changes were observed in the presence of 1-naphthol. Naphthalene, 1-chloronaphthalene, 2-napthol, and 2-naphthoic acid were found to be the mixed inhibitors. Chemical modification studies showed the probable involvement of His, Cys, and Tyr in the binding of 1-naphthol, whereas Trp was found to be involved in the binding of NAD(P)H.

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Abbreviations

MSM:

Minimal salt medium

1-NH:

1-Naphthol 2-hydroxylase

DEPC:

Diethyl pyrocarbonate

NEM:

N-ethyl maleimide

PGO:

Phenylglyoxal

NAI:

N-acetyl imidazole

NBS:

N-bromosuccinimide

TLC:

Thin layer chromatography

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Acknowledgments

Thanks are due to DBT, Govt. of India for research grant to PP and CSIR, Govt. of India for Senior Research fellowship to VDT.

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

    Vikas D. Trivedi, Prabin Majhi & Prashant S. Phale

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  1. Vikas D. Trivedi
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  2. Prabin Majhi
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  3. Prashant S. Phale
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Correspondence to Prashant S. Phale.

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Trivedi, V.D., Majhi, P. & Phale, P.S. Kinetic and Spectroscopic Characterization of 1-Naphthol 2-hydroxylase from Pseudomonas sp. Strain C5. Appl Biochem Biotechnol 172, 3964–3977 (2014). https://doi.org/10.1007/s12010-014-0815-4

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  • DOI: https://doi.org/10.1007/s12010-014-0815-4

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