Partial characterization and three-dimensional structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria
Partial characterization and three-dimensional structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria
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DOI：
10.1046/j.1432-1327.1998.2570001.x
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发表时间：
1998-10-01
期刊：
影响因子：
--
通讯作者：
Flatmark, T
中科院分区：
文献类型：
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作者：
Bjorgo, E;Knappskog, PM;Flatmark, T
苯丙酮尿症患者代谢缺陷的分子基础已被表征为人苯丙氨酸羟化酶（hPAH）基因催化结构域外显子7进化保守基序中的7个错义点突变（R252 G/Q，L255 V/S，A259 V/T和R270 S）和终止突变（G272 X）。突变体在三种异源体外系统中表达，当在大肠杆菌中表达为与麦芽糖结合蛋白的融合蛋白时，五种突变蛋白表现出hPAH折叠和组装为同源四聚体/二聚体的正常能力的缺陷，并且它们大多以无活性的聚集形式回收。仅对于R252 Q和L255 V突变体，回收了催化活性的四聚体和二聚体，对于R252 G，回收了一些二聚体，即各自野生型（wt）形式的活性的20%（R252 Q，四聚体）、44%（L255 V，四聚体）和4.4%（R252 G，二聚体）。当用耦合IR表示时？在体外转录-翻译系统中，所有突变体酶作为具有低同源特异性活性的非磷酸化和磷酸化形式的混合物回收（即，对于L255 V突变体，最大11%的wt-hPAH）。当在人胚肾（A293）细胞中瞬时表达时，尽管PAH mRNA水平正常，但恢复了非常低水平的免疫反应性PAH蛋白。所有这些突变导致变体hPAH蛋白，其揭示了寡聚化缺陷、对体外有限蛋白水解的敏感性增加、细胞稳定性降低以及其催化活性的可变降低。所有这些影响似乎是由于单体的结构扰动，并基于hPAH的催化结构域的晶体结构，提供了一个解释的折叠和单体的寡聚化的突变的影响。
The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems, When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4% (R252G, dimer) of the activity for the respective wild-type (wt) forms. When expressed by a coupled ir? vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e, maximum 11% of wt-hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.