Parales R E, Emig M D, Lynch N A, Gibson D T
Department of Microbiology and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City 52242, USA.
J Bacteriol. 1998 May;180(9):2337-44. doi: 10.1128/JB.180.9.2337-2344.1998.
Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.
细菌三组分双加氧酶系统由还原酶和铁氧化还原蛋白组分组成，它们将电子从NAD(P)H传递给末端加氧酶。在大多数情况下，加氧酶由两个不同的亚基（α和β）组成。为了评估加氧酶的α和β亚基对底物特异性的贡献，通过在大肠杆菌中共表达来自两个相容质粒的基因来形成杂合双加氧酶。用四种底物（吲哚、萘、2,4-二硝基甲苯和2-硝基甲苯）测试了含有四种不同β亚基的杂合萘和2,4-二硝基甲苯双加氧酶的活性。在有活性的杂合体中，小亚基的替换影响产物形成速率，但对测试底物的底物范围、区域特异性或氧化产物的对映体纯度没有影响。这些研究表明，加氧酶的小亚基对活性至关重要，但在决定这些酶的特异性方面不发挥主要作用。
