Leopoldseder Sonja, Claren Jörg, Jürgens Catharina, Sterner Reinhard
Universität zu Köln, Institut für Biochemie, Otto-Fischer-Strasse 12-14, D-50674 Köln, Germany.
J Mol Biol. 2004 Apr 2;337(4):871-9. doi: 10.1016/j.jmb.2004.01.062.
The (betaalpha)(8)-barrel enzymes N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (tHisA) and imidazole glycerol phosphate synthase (tHisF) from Thermotoga maritima catalyze two successive reactions in the biosynthesis of histidine. In both enzymes, aspartate residues at the C-terminal end of beta-strand 1 (Asp8 in tHisA and Asp11 in tHisF) and beta-strand 5 (Asp127 in tHisA and Asp130 in tHisF) are essential for catalytic activity. It was demonstrated earlier that in tHisA the substitution of Asp127 by valine (tHisA-D127V) generates phosphoribosylanthranilate isomerase (TrpF) activity, a related (betaalpha)(8)-barrel enzyme participating in tryptophan biosynthesis. It is shown here that in tHisF the corresponding substitution of Asp130 by valine (tHisF-D130V) also generates TrpF activity. To determine the effectiveness of individual amino acid exchanges in these conversions, each of the 20 standard amino acid residues was introduced at position 127 of tHisA and 130 of tHisF by saturation random mutagenesis. The tHisA-D127X and tHisF-D130X variants with TrpF activity were identified by selection in vivo, and the proteins purified and characterized. The results obtained show that removal of the negatively charged carboxylate side-chain at the C-terminal end of beta-strand 5 is sufficient to establish TrpF activity in tHisA and tHisF, presumably because it allows the binding of the negatively charged TrpF substrate, phosphoribosylanthranilate. In contrast, the double mutants tHisA-D8N+D127V and tHisF-D11N+D130V did not show detectable activity, demonstrating that the aspartate residues at the C-terminal end of beta-strand 1 are essential for catalysis of the TrpF reaction. The ease with which TrpF activity can be established on both the tHisA and tHisF scaffolds supports the evolutionary relationship of these three enzymes and highlights the functional plasticity of the (betaalpha)(8)-barrel enzyme fold.
来自嗜热栖热菌的（βα）8桶状酶N'-[（5'-磷酸核糖基）甲脒基]-5-氨基咪唑-4-甲酰胺核糖核苷酸异构酶（tHisA）和咪唑甘油磷酸合酶（tHisF）催化组氨酸生物合成中的两个连续反应。在这两种酶中，β链1（tHisA中的Asp8和tHisF中的Asp11）和β链5（tHisA中的Asp127和tHisF中的Asp130）C末端的天冬氨酸残基对催化活性至关重要。早期已证明，在tHisA中用缬氨酸取代Asp127（tHisA-D127V）会产生磷酸核糖基邻氨基苯甲酸异构酶（TrpF）活性，TrpF是一种参与色氨酸生物合成的相关（βα）8桶状酶。本文表明，在tHisF中用缬氨酸相应取代Asp130（tHisF-D130V）也会产生TrpF活性。为了确定这些转化中单个氨基酸交换的有效性，通过饱和随机诱变将20种标准氨基酸残基分别引入tHisA的第127位和tHisF的第130位。具有TrpF活性的tHisA-D127X和tHisF-D130X变体通过体内筛选鉴定，然后对纯化的蛋白质进行表征。所得结果表明，去除β链5 C末端带负电荷的羧基侧链足以在tHisA和tHisF中建立TrpF活性，推测这是因为它允许带负电荷的TrpF底物磷酸核糖基邻氨基苯甲酸结合。相比之下，双突变体tHisA-D8N+D127V和tHisF-D11N+D130V未显示出可检测的活性，表明β链1 C末端的天冬氨酸残基对TrpF反应的催化至关重要。在tHisA和tHisF支架上都能轻松建立TrpF活性，这支持了这三种酶的进化关系，并突出了（βα）8桶状酶折叠的功能可塑性。
