Biochemical and molecular characterization of 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7

1-Hydroxy-2-naphthoate dioxygenase, which cleaves the singly hydroxylated aromatic ring, tvas 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 K-m and k(cat) values of this enzyme for 1-hydroxy-2-naphthoate were 10 mu M 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 or 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(lI) 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.