Abstract：
L-tryptophan (l-Trp), a vital amino acid for the survival of various organisms, is synthesized by the enzyme tryptophan synthase (TS) in organisms such as eubacteria, archaebacteria, protista, fungi, and plantae. TS, a pyridoxal 5\'-phosphate (PLP)-dependent enzyme, comprises α and β subunits that typically form an α2β2 tetramer. The enzyme\'s activity is regulated by the conformational switching of its α and β subunits between the open (T state) and closed (R state) conformations. Many microorganisms rely on TS for growth and replication, making the enzyme and the l-Trp biosynthetic pathway potential drug targets. For instance, Mycobacterium tuberculosis, Chlamydiae bacteria, Streptococcus pneumoniae, Francisella tularensis, Salmonella bacteria, and Cryptosporidium parasitic protozoa depend on l-Trp synthesis. Antibiotic-resistant salmonella strains have emerged, underscoring the need for novel drugs targeting the l-Trp biosynthetic pathway, especially for salmonella-related infections. A single amino acid mutation can significantly impact enzyme function, affecting stability, conformational dynamics, and active or allosteric sites. These changes influence interactions, catalytic activity, and protein-ligand/protein-protein interactions. This study focuses on the impact of mutating the βGln114 residue on the catalytic and allosteric sites of TS. Extensive molecular dynamics simulations were conducted on E(PLP), E(AEX1), E(A-A), and E(C3) forms of TS using the WT, βQ114A, and βQ114N versions. The results show that both the βQ114A and βQ114N mutations increase protein backbone root mean square deviation fluctuations, destabilizing all TS forms. Conformational and hydrogen bond analyses suggest the significance of βGln114 drifting away from cofactor/intermediates and forming hydrogen bonds with water molecules necessary for l-Trp biosynthesis. The βQ114A mutation creates a gap between βAla114 and cofactor/intermediates, hindering hydrogen bond formation due to short side chains and disrupting β-sites. Conversely, the βQ114N mutation positions βAsn114 closer to cofactor/intermediates, forming hydrogen bonds with O3 of cofactors/intermediates and nearby water molecules, potentially disrupting the l-Trp biosynthetic mechanism.
摘要：
L-色氨酸(l-Trp),一种对各种生物生存至关重要的氨基酸，是由细菌等生物体中的色氨酸合成酶（TS）合成的，古细菌,protista,真菌,和植物。TS,吡哆醛5'-磷酸（PLP）依赖性酶，包含通常形成α2β2四聚体的α和β亚基。酶的活性受到其α和β亚基在开放（T状态）和封闭（R状态）构象之间的构象转换的调节。许多微生物依靠TS进行生长和复制，使酶和l-Trp生物合成途径成为潜在的药物靶标。例如,结核分枝杆菌,衣原体细菌，肺炎链球菌,图拉西斯,沙门氏菌,隐孢子虫寄生原虫依赖于l-Trp合成。出现了耐抗生素的沙门氏菌菌株,强调了对靶向l-Trp生物合成途径的新药的需求，尤其是沙门氏菌相关的感染。单个氨基酸突变可以显著影响酶的功能，影响稳定性，构象动力学，和活性或变构位点。这些变化影响相互作用，催化活性，和蛋白质-配体/蛋白质-蛋白质相互作用。这项研究的重点是突变βGln114残基对TS的催化和变构位点的影响。在E(PLP)上进行了广泛的分子动力学模拟，E(AEX1),E(A-A),和使用WT的TS的E(C3)形式，βQ114A,和βQ114N版本。结果表明,βQ114A和βQ114N突变均增加蛋白骨架均方根偏差波动,破坏所有TS形式的稳定性。构象和氢键分析表明βGln114远离辅因子/中间体并与l-Trp生物合成所需的水分子形成氢键的重要性。βQ114A突变在βAla114和辅因子/中间体之间产生缺口，由于短侧链和破坏β位点而阻碍氢键形成。相反，βQ114N突变位置βAsn114更接近辅因子/中间体，与辅助因子/中间体的O3和附近的水分子形成氢键，可能破坏l-Trp生物合成机制。
