To achieve a high yield of terpenoid-based therapeutics, 1-deoxy-d-xylulose-5-phosphate (DXP) pathway has been significantly exploited for the production of downstream enzymes. The DXP synthase (DXS) enzyme, the initiator of this pathway, is pivotal for the convergence of carbon flux, and is computationally studied well for the industrially utilized generally regarded as safe (GRAS) bacterium Bacillus subtilis to decode its vital regions for aiding the construction of a functionally improved mutant library. For the 546 sequence dataset of DXS sequences, a representative set of 108 sequences is created, and it shows a significant evolutionary divergence across different species clubbed into 37 clades, whereas three clades are observed for the 76 sequence dataset of Bacillus subtilis. The DXS enzyme, sharing a statistically significant homology to transketolase, is shown to be evolutionarily too distant. By the mutual information-based co-evolutionary network and hotspot analysis, the most crucial loci within the active site are deciphered. The 650-residue representative structure displays a complete conservation of 114 loci, and only two co-evolving residues ASP154 and ILE371 are found to be the conserved ones. Lastly, P318D is predicted to be the top-ranked mutation causing the increase in the thermodynamic stability of 6OUW. The study excavates the vital functional, phylogenetic, and conserved residues across the active site of the DXS protein, the key rate-limiting controller of the entire pathway. It would aid to computationally understand the evolutionary landscape of this industrially useful enzyme and would allow us to widen its substrate repertoire to increase the enzymatic yield of unnatural molecules for in vivo and in vitro applications.

中文翻译：

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通过探索枯草芽孢杆菌的DXS同源物挖掘功能关键的活性位点残基

为了获得高产率的基于萜类的治疗剂，已广泛地利用1-脱氧-d-木酮糖-5-磷酸（DXP）途径生产下游酶。DXP合酶（DXS）酶是该途径的发起者，对于碳通量的收敛起关键作用，并且在工业上被普遍认为是安全的（GRAS）枯草芽孢杆菌细菌用于对其辅助生命的关键区域进行解码的计算研究良好。功能上改进的突变体文库的构建。对于DXS序列的546个序列数据集，创建了108个序列的代表性集合，它显示了分成37个进化枝的不同物种之间的显着进化差异，而对于枯草芽孢杆菌的76个序列数据集，观察到了3个进化枝。DXS酶 与转酮醇酶具有统计学上显着的同源性，在进化上距离太远。通过基于互信息的协同进化网络和热点分析，可以了解活动站点内最关键的基因座。650个残基的代表性结构显示了114个基因座的完整保守性，只有两个共同进化的残基ASP154和ILE371被发现是保守的。最后，P318D被认为是导致6OUW热力学稳定性增加的最高突变。该研究挖掘了DXS蛋白活性位点的重要功能，系统发育和保守残基，而DXS蛋白是整个途径的关键限速控制器。