title: Kinetic and spectroscopic characterization of 1-naphthol 2-hydroxylase from Pseudomonas sp. strain C5.
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.
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, 400 076, India.
authors.forename: Vikas D
authors.initials: VD
authors.lastname: Trivedi
authors.forename: Prabin
authors.initials: P
authors.lastname: Majhi
authors.forename: Prashant S
authors.initials: PS
authors.lastname: Phale
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013057:Spectrum Analysis; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C519715:1-naphthol-2-hydroxylase, Pseudomonas; D006899:Mixed Function Oxygenases