Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia

T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.     

基于结构域活性分析的大肠杆菌T蛋白结构研究

大肠杆菌T蛋白含有三个结构域:分支酸变位酶、预苯酸脱氢酶和调节结构域。文章作者分段克隆了T蛋白的分支酸变位酶、预苯酸脱氢酶和调节结构域等片段,并对其进行了活性研究。研究发现,定位于N末端的分支酸变位酶结构域的比活性虽然不高,而稳定性较好;同时拥有调节结构域和预苯酸脱氢酶结构域的C末端片段,其预苯酸脱氢酶比活性的剩余百分率虽然高于分支酸变位酶结构域,但稳定性较差。作者进而表达了C末端切除38个氨基酸的T/1-336片段,发现预苯酸脱氢酶活性彻底丧失,而其分支酸变位酶和调节结构域的活性却基本保留。这说明T蛋白中分支酸变位酶结构域拥有一个相对独立、完整的结构,而预苯酸脱氢酶结构域和调节结构域交织共存,结构松散。     

T-protein of the glycine decarboxylase multienzyme complex: evidence for partial similarity to formyltetrahydrofolate synthetase

We have isolated and sequenced cDNA clones encoding T-protein of the glycine decarboxylase complex from three plant species, Flaveria pringlei, Solanum tuberosum and Pisum sativum. The predicted amino acid sequences of these clones are at least 87% identical and all are similar to the predicted sequences of the bovine, human, chicken and Escherichia coli T-proteins. Alignment of all these sequences revealed conserved domains, one of which showed a significant similarity to a part of the formyltetrahydrofolate synthetases from procaryotes and eucaryotes. This suggests that the T-protein sequence is not as unique as previously thought.     

大肠杆菌P蛋白和T蛋白的调节结构域互换研究

在细菌、真菌及植物中,分支酸是一种位于关键分叉点上的中间代谢物,是所有芳香族氨基酸合成的共同前体.它可在双功能酶分支酸变位酶(CM)和预苯酸脱水酶(PDT)的催化下合成苯丙氨酸,在另一个双功能酶分支酸变位酶和预苯酸脱氢酶(PDH)的催化下合成酪氨酸.前者被称为P蛋白,后者被称为T蛋白.大肠杆菌P蛋白和T蛋白有着类似的结构,P蛋白由CMp、PDT和调节结构域３个独立结构域组成,其变构调节因子是苯丙氨酸.T蛋白只有CMt和PDH两个独立结构域组成,起变构调节作用的调节结构域与PDH密不可分,其变构调节因子是酪氨酸.为了研究P蛋白和T蛋白的调节结构域的变构调节作用,应用融合蛋白技术将P蛋白和T蛋白的调节结构域进行了互换.结果发现,互换了的调节结构域仍然具有变构调节作用,而且调节结构域的互换导致了变构调节因子的互换,说明调节结构域对酶活性的调节作用是非专一的,而其R结构域与调节因子的结合却是专一的.     

利用蛋白质限制性水解法解析大肠杆菌T蛋白的独立结构域

为了解析分支酸变位酶和预苯酸脱氢酶在大肠杆菌T蛋白的定位,根据T蛋白限制性水解结果,分段克隆分支酸变位酶和预苯酸脱氢酶.T蛋白限制性水解结果显示,第93位氨基酸是大片段的N端,分段克隆的1～93 片段测定得到分支酸变位酶活性,96～373片段得到了预苯酸脱氢酶活性.研究表明,大肠杆菌T蛋白由两个独立结构域组成,N端93个氨基酸组成了分支酸变位酶,C端277个氨基酸组成了预苯酸脱氢酶.