Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase -: A small subunit, HpdC, is essential for catalytic activity
Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase -: A small subunit, HpdC, is essential for catalytic activity
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DOI：
10.1111/j.1432-1033.2004.04152.x
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发表时间：
2004-06-01
期刊：
影响因子：
--
通讯作者：
Selmer, T
中科院分区：
文献类型：
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作者：
Andrei, PI;Pierik, AJ;Selmer, T
艰难梭菌产生的对羟基苯乙酸酯脱羧酶催化对羟基苯乙酸酯脱羧基生成细胞毒性化合物对甲酚。编码甘氨酰自由基酶和活化酶两个亚基的三个基因已被克隆并在大肠杆菌中表达。将重组酶用于构建体外催化功能系统。从艰难梭菌中纯化的脱羧酶是一种几乎没有活性的同源二聚体蛋白(β(2))，而重组酶是一种具有催化活性的异八聚体(β(4)伽马(4))复合体，可被艰难梭菌的内源活化酶或重组活化酶激活，比活力为7U mg(-1)。初步结果表明，小亚基的磷酸化是寡聚体状态改变的原因。这些数据指向脱羧酶小亚基的一个基本功能，并可能表明该系统的独特调节特性。
p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (beta(2)), the recombinant enzyme was a hetero-octameric (beta(4)gamma(4)), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U.mg(-1). Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system.