Iwabuchi T, Harayama S
Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026, Japan.
J Biol Chem. 1998 Apr 3;273(14):8332-6. doi: 10.1074/jbc.273.14.8332.
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.
从降解菲的诺卡氏菌属菌株KP7中纯化出了1-羟基-2-萘酸双加氧酶，该酶可裂解单羟基化的芳香环。经SDS-聚丙烯酰胺凝胶电泳测定，纯化后的酶分子量为45 kDa，经凝胶过滤色谱测定为270 kDa。该酶对1-羟基-2-萘酸的表观Km值和kcat值分别为10 μM和114 s-1。1摩尔1-羟基-2-萘酸被氧化时消耗1摩尔分子氧。该酶每摩尔亚基含有1摩尔Fe(II)，并被邻菲罗啉灭活。用邻菲罗啉灭活的酶通过与硫酸亚铁和抗坏血酸孵育而复活。因此，该酶表现出活性需要Fe(II)。从黏粒文库中筛选出该酶的结构基因并进行测序，1-羟基-2-萘酸基因长度为1161个碱基对。该酶推导的氨基酸序列与其他裂解双羟基化芳香环的环裂解双加氧酶的序列不同。
