The obligately anaerobic, denitrifying bacterium Azoarcus anaerobius strain LuFRes1 grows with resorcinol (1,3-dihydroxybenzene) as sole carbon and energy source. Resorcinol is oxidized to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by resorcinol hydroxylase (RH), an inducible membrane-bound enzyme. Sequence comparison places resorcinol hydroxylase into the group of anaerobic molybdopterin oxidoreductases and dimethyl sulfoxide reductase-like enzymes. In the large subunit, a molybdopterin-binding domain was predicted, and the small subunit most likely contains two [4Fe-4S] centers. Growth of molybdate-starved cells was inhibited by tungstate, and in vitro resorcinol hydroxylase activity was inhibited by arsenite and selenite that are known to inhibit molybdenum-containing enzymes. The two genes encoding resorcinol hydroxylase could be expressed in Escherichia coli but the products remained in inclusion bodies. All attempts to purify RH from A. anaerobius or to produce soluble, active RH in E. coli failed. Nevertheless, RH was produced as a C-terminally Strep-tagged protein from plasmid pSKM1 in Thauera aromatica AR1 transconjugants carrying a transposon insertion in the coding gene for the large (ΔrhL) or the small subunit (ΔrhS) of RH from cosmid R+. RH in the membrane fraction of wild-type transconjugant T. aromatica AR1/R+ showed a specific activity of 80 mU mg-1, and the specific activity of RH in the membranes of the complemented mutants was in the same range (80-95 mU mg-1). We conclude that RH of A. anaerobius is a membrane-bound molybdoenzyme consisting of two subunits which might require a further loosely bound subunit as membrane anchor.

中文翻译：

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Azoarcus anaerobius 的间苯二酚羟化酶：钼依赖性、活性和异源表达

专性厌氧反硝化细菌 Azoarcus anaerobius 菌株 LuFRes1 以间苯二酚（1,3-二羟基苯）作为唯一碳源和能源生长。间苯二酚被间苯二酚羟化酶 (RH)（一种诱导膜结合酶）氧化为羟基氢醌（1,2,4-三羟基苯）。序列比较将间苯二酚羟化酶归入厌氧钼蝶呤氧化还原酶和二甲亚砜还原酶样酶组。在大亚基中，预测了钼蝶呤结合域，小亚基很可能包含两个 [4Fe-4S] 中心。钨酸盐会抑制钼酸盐饥饿细胞的生长，并且已知抑制含钼酶的亚砷酸盐和亚硒酸盐会抑制体外间苯二酚羟化酶活性。编码间苯二酚羟化酶的两个基因可以在大肠杆菌中表达，但产物保留在包涵体中。从厌氧菌中纯化 RH 或在大肠杆菌中产生可溶性活性 RH 的所有尝试都失败了。尽管如此，RH 是从质粒 pSKM1 中产生的作为 C 末端链球菌标记的蛋白质在 Thauera 芳香族 AR1 转接合子中携带转座子插入到来自粘粒 R+ 的 RH 的大 (ΔrhL) 或小亚基 (ΔrhS) 的编码基因中。野生型芳香木霉AR1/R+膜组分中RH的比活为80 mU mg-1，互补突变体膜中RH的比活在相同范围内（80-95 mU毫克-1）。我们得出结论，A 的 RH。