Functional domains of human tryptophan hydroxylase 2 (hTPH2)
Functional domains of human tryptophan hydroxylase 2 (hTPH2)
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DOI：
10.1074/jbc.m602817200
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发表时间：
2006-09-22
影响因子：
4.8
通讯作者：
Vrana, Kent E.
中科院分区：
文献类型：
--
作者：
Carkaci-Salli, Nurgul;Flanagan, John M.;Vrana, Kent E.
色氨酸羟化酶（TPH）是5-羟色胺生物合成的限速酶。一个新的基因，称为TPH 2，最近被描述。该基因优先在中枢神经系统中表达，而原始TPH 1是外周基因。我们在大肠杆菌中表达了人色氨酸羟化酶2（hTPH 2）和两个缺失突变体（N Delta 150和N Delta 150/C Delta 24），使用异丙基β-D-硫代半乳糖苷的自诱导。该表达系统产生具有相对低溶解度的活性野生型TPH 2。缺乏NH 2-末端调节结构域的突变体的溶解度增加。hTPH 2、N Delta 150和N Delta 150/C Delta 24的溶解度分别为6.9%、62%和97.5%。去除调节结构域也使酶的稳定性增加了6倍以上（t（1/2），37 ℃）。野生型hTPH 2，像芳香族氨基酸羟化酶超家族的其他成员一样，以同源四聚体（在尺寸排阻色谱上为236 kDa）存在。类似地，N Delta 150也作为四聚体（168 kDa）迁移。相反，去除NH 2-末端结构域和COOH-末端，推定的亮氨酸拉链四聚化结构域产生单体酶（39 kDa）。有趣的是，去除NH 2-末端调节结构域并不影响任一底物的米氏常数，但确实增加了Vmax值。这些数据确定的NH 2-末端调节结构域作为来源的hTPH 2的不稳定性和降低的溶解度。
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (N Delta 150 and N Delta 150/C Delta 24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH2-terminal regulatory domain. The solubility of hTPH2, N Delta 150, and N Delta 150/C Delta 24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t(1/2) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer ( 236 kDa on size exclusion chromatography). Similarly, N Delta 150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH2-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme ( 39 kDa). Interestingly, removal of the NH2-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V-max values. These data identify the NH2-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.