Biochemical and Molecular Characterization of 1-Hydroxy-2-naphthoate Dioxygenase from Nocardioides sp. KP7

Tokuro Iwabuchi,Shigeaki Harayama

doi:10.1074/jbc.273.14.8332

Abstract

1-Hydroxy-2-naphthoate dioxygenase, which cleaves the singly hydroxylated aromatic ring, was purified from phenanthrene-degrading Nocardioides sp. strain KP7. The purified enzyme had a molecular mass of 45 kDa by SDS-polyacrylamide gel electrophoresis and 270 kDa by gel filtration chromatography. The apparent Km and kcat values of this enzyme for 1-hydroxy-2-naphthoate were 10 microM and 114 s-1, respectively. One mole of molecular oxygen was consumed when 1 mol of 1-hydroxy-2-naphthoate was oxidized. This enzyme contained 1 mol of Fe(II)/mol of the subunit and was inactivated by o-phenanthroline. The enzyme that had been inactivated by o-phenanthroline was reactivated by incubating with FeSO4 and ascorbic acid. Thus, Fe(II) was required for the enzyme to exhibit activity. The structural gene for this enzyme was screened from a cosmid library and then sequenced, the length of the 1-hydroxy-2-naphthoate gene being 1161 base pairs. The deduced amino acid sequence of this enzyme was different from those of other ring-cleaving dioxygenases that cleave the doubly hydroxylated aromatic ring.

Highlights

Dioxygenases cleaving an aromatic ring that possesses two adjacent hydroxyl groups are divided into two classes
In the case of gentisate containing two hydroxyl groups at a position para to each other, the ring is cleaved between carbon 1 and carbon 2 by gentisate 1,2-dioxygenase, and the ring-fission mechanism is believed to resemble that of extradiol-type fission [4, 5]
A model for the mechanism of extradiol dioxygenases has recently been proposed, and it was suggested that two hydroxyl groups of the substrate would interact with the catalytic iron, deprotonation of one of these hydroxyl groups being the first step of ring cleavage [16]. 1-Hydroxy-2-naphthoate dioxygenase is unique among ring-cleaving dioxygenases because it can cleave a singly hydroxylated aromatic ring

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strain—Nocardioides sp. strain KP7, which degrades phenanthrene via o-phthalate [11], was used in this study. The 1-hydroxy-2-naphthoate dioxygenase activity was recovered in the supernatant fluid This supernatant was passed through a Millex-GV filter (0.45 ␮m pore size; Millipore) and loaded into a hydrophobic interaction column (TSKgel phenyl-5PW, 21.5 ϫ 150 mm; Tosoh) that had been pre-equilibrated with a 20 mM Tris-H2SO4 buffer (pH 7.5) containing 0.6 M ammonium sulfate. Gene Cloning and Sequencing—Two degenerate PCR1 primers (the N primer and cC primer), which could collaboratively amplify a 75-bp-long DNA fragment encoding the amino-terminal 25 amino acid residues of 1-hydroxy-2-naphthoate dioxygenase, were designed. Pooled fractions containing active 1-hydroxy-2-naphthoate dioxygenase were concentrated to 80 ␮l in a 1.5-ml centrifuge tube fitted with an Ultrafree C3-LGC membrane (Millipore), loaded into a gel filtration column (TSKgel G3000SWXL, 7.8 ϫ 300 mm; Tosoh), and eluted with a mobile phase of 20 mM Tris-H2SO4 (pH 6.8) containing 100 mM Na2SO4 at a flow rate of 0.5 ml minϪ1. Amino-terminal Sequencing—The amino-terminal sequence of the purified enzyme was determined by Edman degradation with an automated protein sequencer (Perkin-Elmer model 477)

RESULTS

Total activity

DISCUSSION