Structure-function relationship of novel lysine biosynthetic enzymes and analysis of molecular evolution of the pathway

1)对嗜热栖热菌高乌头酸酶的分析表明，该酶催化第3步反应，但不催化第2步反应。2）测定了α-氨基己二酸氨基转移酶（AAA-AT）的几种形态的X-射线结构。AAA-AT不仅识别赖氨酸生物合成中的中间体2-氧代异己酸，而且识别亮氨酸和苯丙氨酸生物合成中的相应化合物。X-射线衍射结构表明，N-末端螺旋结构的移动的是该酶具有广泛底物特异性的原因，而螺旋结构上的精氨酸残基对该酶的催化功能起着至关重要的作用。3）通过DNA改组技术，高异柠檬酸脱氢酶被成功地转化为利用2-异丙基苹果酸的酶，2-异丙基苹果酸是亮氨酸生物合成中的相应化合物，但不被野生型高异柠檬酸脱氢酶识别为底物。分析所得到的进化酶和每个氨基酸取代的效果表明，在进化酶中引入的所有取代对底物特异性的变化具有积极的影响。4)LysX可能是对赖氨酸生物合成途径中α-氨基己二酸的α-氨基进行修饰。为了阐明修饰的机理，将来自嗜热嗜热菌HB 27的LysX及其在东京硫化叶菌中的寄生菌结晶。5)赖氨酸通过类似于大肠杆菌trp操纵子的减毒机制调控赖氨酸生物合成主基因簇的转录。此外，转录还受精氨酸生物合成中的转录调节因子ArgR和脯氨酸代谢中的转录调节因子PutR的调节。

1)Detailed analysis of homoaconitase from Thermus thermophilus revealed that the enzyme catalyzes the 3rd reaction but could not the 2nd reaction of the pathway. 2)X-ray structures of α-aminoadipate aminotransferase (AAA-AT) were determined in the several forms. AAA-AT recognizes not only 2-oxoisocaproate, an intermediate in lysine biosynthesis, but also the corresponding compounds in the leucine and phenylalanine biosyntheses. The X-ray structure indicates that the mobile at helix located N-terminus is responsible for the broad substrate specificity of this enzyme, and that an arginine residue on the helix plays a crucial role in the catalytic function. 3)By DNA shuffling technique, homoisocitrate dehydrogenase was successfully converted to the enzyme that utilizes 2-isopropylmalate that is the corresponding compound in leucine biosynthesis but is not recognized as a substrate by wild-type homoisocitrate dehydrogenase. Analysis of the resulting evolved enzyme and effect of each amino acid substitution revealed that all the replacements introduced in the evolved enzyme had positive effect on the change in the substrate specificity. 4)LysX is suggested to modify α-amino group of α-aminoadipate in lysine biosynthetic pathway. To elucidate the mechanism for the modification, LysX from T.thermophilus HB27 and its paralogue in Sulfolobus tokodaii were crystallized. 5)Transcription of major gene cluster for lysine biosynthesis is regulated by lysine through attenuation mechanism similar to that of trp operon in Escherichia coli. In addition, the transcription is further regulated by ArgR, a transcriptional regulator in arginine biosynthesis, and PutR, a transcriptional regulator in proline metabolism.