Abstract：
Mycobacterium tuberculosis enolase is an essential glycolytic enzyme that catalyzes the conversion of 2, phosphoglycerate (PGA) to phosphoenol pyruvate (PEP). It is also a crucial link between glycolysis and the tricarboxylic acid (TCA) pathway. The depletion of PEP has recently been associated with the emergence of non-replicating drug resistant bacteria. Enolase is also known to exhibit multiple alternate functions, such as promoting tissue invasion via its role as a plasminogen (Plg) receptor. In addition, proteomic studies have identified the presence of enolase in the Mtb degradosome and in biofilms. However, the precise role in these processes has not been elaborated. The enzyme was recently identified as a target for 2-amino thiazoles - a novel class of anti-mycobacterials. In vitro assays and characterization of this enzyme were unsuccessful due to the inability to obtain functional recombinant protein. In the present study, we report the expression and characterization of enolase using Mtb H37Ra as a host strain. Our study demonstrates that the enzyme activity and alternate functions of this protein are significantly impacted by the choice of expression host (Mtb H37Ra or E. coli). Detailed analysis of the protein from each source revealed subtle differences in the post-translational modifications. Lastly, our study confirms the role of enolase in Mtb biofilm formation and describes the potential for inhibiting this process.
摘要：
结核分枝杆菌烯醇化酶是一种必需的糖酵解酶，可催化2，磷酸甘油酸（PGA）转化为磷酸烯醇丙酮酸（PEP）。它也是糖酵解和三羧酸(TCA)途径之间的关键联系。PEP的消耗最近与非复制耐药细菌的出现有关。还已知烯醇化酶表现出多种替代功能，例如通过其作为纤溶酶原（Plg）受体的作用促进组织侵入。此外,蛋白质组学研究已经确定了烯醇化酶在Mtb降解体和生物膜中的存在。然而,在这些过程中的确切作用尚未详细说明。该酶最近被确定为2-氨基噻唑的靶标-一种新型的抗分枝杆菌。由于无法获得功能性重组蛋白，因此该酶的体外测定和表征不成功。在本研究中,我们报道了使用MtbH37Ra作为宿主菌株的烯醇化酶的表达和表征。我们的研究表明，该蛋白质的酶活性和替代功能受到表达宿主（MtbH37Ra或大肠杆菌）选择的显着影响。对来自每个来源的蛋白质的详细分析揭示了翻译后修饰的细微差别。最后,我们的研究证实了烯醇化酶在Mtb生物膜形成中的作用，并描述了抑制这一过程的潜力。
