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

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Title Kinetic and Spectroscopic Characterization of 1-Naphthol 2-hydroxylase from Pseudomonas sp Strain C5
 
Creator TRIVEDI, VD
MAJHI, P
PHALE, PS
 
Subject 1-Naphthol 2-hydroxylase
Flavoenzyme
Hydroxylation efficiency
Nonsubstrate effector
Spectroscopic characterization
Pseudomonas
Carbaryl metabolism
PARA-HYDROXYBENZOATE HYDROXYLASE
CONFORMATIONAL-CHANGE
PHENOL HYDROXYLASE
PURIFICATION
METABOLISM
SUBSTRATE
CARBARYL
PUTIDA
1,2-DIHYDROXYNAPHTHALENE
FLUORESCENS
 
Description 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 (similar to 30 %) followed by 4-chloro 1-naphthol (similar to 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 mu M) and salicylate (K (d) = 1027 mu M). 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.
 
Publisher HUMANA PRESS INC
 
Date 2014-12-28T14:28:50Z
2014-12-28T14:28:50Z
2014
 
Type Article
 
Identifier APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 172(8)3964-3977
0273-2289
1559-0291
http://dx.doi.org/10.1007/s12010-014-0815-4
http://dspace.library.iitb.ac.in/jspui/handle/100/16754
 
Language English