Hopper D J, Kaderbhai M A
Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, Wales, U.K.
Biochem J. 1999 Dec 1;344 Pt 2(Pt 2):397-402.
2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) was purified to homogeneity from Alcaligenes sp. 4HAP grown on 4-hydroxyacetophenone. Measurements of the M(r) of the native enzyme ranged from 81600 to 87000, whereas values of 21000 and 20379 were given by SDS/PAGE and electrospray MS respectively. The enzyme is a homotetramer and contains one atom of iron per molecule of enzyme. From C- and N-terminal analyses, primers for PCR were designed and the dad gene cloned and sequenced. The predicted amino acid sequence of dad, deduced from the nucleotide sequence, confirms the N-terminal amino acid sequencing data and contains the sequence of an internal tryptic peptide. It gave a calculated M(r) of 20364. The gene was expressed in Escherichia coli and yielded active enzyme. The derived amino acid sequence does not show significant similarity to other dioxygenases or any strong similarity to protein sequences presently available in the databases.
从以4-羟基苯乙酮为生长底物的产碱杆菌属菌株4HAP中纯化得到了2,4'-二羟基苯乙酮双加氧酶（EC 1.13.11.41），使其达到了均一状态。对天然酶的相对分子质量测定结果在81600至87000之间，而十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）和电喷雾质谱分别给出的数值为21000和20379。该酶是同四聚体，每个酶分子含有一个铁原子。通过C端和N端分析，设计了用于聚合酶链反应（PCR）的引物，并对dad基因进行了克隆和测序。根据核苷酸序列推导得到的dad基因预测氨基酸序列，证实了N端氨基酸测序数据，并且包含一个内部胰蛋白酶肽段的序列。其计算得到的相对分子质量为20364。该基因在大肠杆菌中表达并产生了活性酶。推导得到的氨基酸序列与其他双加氧酶没有显著的相似性，与数据库中目前可用的蛋白质序列也没有任何强烈的相似性。
