Enya Mayu, Aoyagi Keiko, Hishikawa Yoshihiro, Yoshimura Azusa, Mitsukura Koichi, Maruyama Kiyofumi
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Biosci Biotechnol Biochem. 2012;76(3):567-74. doi: 10.1271/bbb.110867.
The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 µM and 6.28 µmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.
从伯克霍尔德菌属菌株AZ11中克隆到了编码2,4'-二羟基苯乙酮（DHAP）双加氧酶的基因dad。将起始密码子GTG转换为ATG以在大肠杆菌中实现该酶的高水平表达。该酶具有适度的热稳定性，对重组酶进行了简单纯化。该酶（M(r)=90 kDa）是一种同四聚体，亚基M(r)为23 kDa。它含有1.69摩尔的非血红素铁，呈深灰色。在厌氧条件下将其与DHAP一起孵育时，由于形成了酶-DHAP复合物，400 nm左右的吸光度增加。多序列比对表明，杯形结构域中的His77、His79、His115和Glu96可能是金属配体。DHAP的表观K(m)和表观V(max)分别估计为1.60 μM和6.28 μmol/min/mg。2-羟基苯乙酮是一种较差的底物。CuCl(2)和HgCl(2)强烈抑制该酶，而FeSO(4)对其有微弱的激活作用。
