Dzierszinski F, Mortuaire M, Dendouga N, Popescu O, Tomavo S
Laboratoire de Chimie Biologique, Centre National de la Recherche Scientifique UMR N(o)8576, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq cedex, 59655, France
J Mol Biol. 2001 Jun 22;309(5):1017-27. doi: 10.1006/jmbi.2001.4730.
The precise molecular mechanisms underlying the switch between the two developmental stages of Toxoplasma gondii, and the metabolic adaptations occurring during this stage conversion are poorly understood. Because inhibitors of mitochondrial respiration are known to trigger differentiation from tachyzoite into bradyzoite stages, we believe that some of the switch components may be sought in the regulation of central carbohydrate metabolism. We have previously described a cDNA encoding a bradyzoite-specific enolase, ENO1. We now report the isolation and characterization of another enolase-encoding cDNA (ENO2) that is expressed preferentially in the tachyzoite stage. The deduced amino acid sequences of ENO1 and ENO2 share 73.65 % identity. They both display significant homologies to plant enolases with the presence of two plant-like peptide insertions, a pentapeptide EWGW(Y)C(S) and a dipeptide EK (or DK). We demonstrate that deletions of the ENO1 pentapeptide motif on its own or together with the dipeptide reduce drastically the affinity for the 2PGA substrate, suggesting that the evolutionary acquisition of these peptides in enolases of land plants and apicomplexan parasites contribute a specific function to their enzymatic activities. T. gondii ENO1 and ENO2 were also expressed as active recombinant enzymes in Escherichia coli. While ENO1 and ENO2 display similar K(m) values, the pure tachyzoite-specific enzyme (ENO2) has a threefold specific activity at V(max) compared with that of the bradyzoite-specific enolase (ENO1). Moreover, immunoblot analyses performed using polyclonal antibodies raised against the recombinant enzymes revealed that the native enolase in tachyzoite and bradyzoite are also antigenically distinct. Taken together, our results indicate that the differences witnessed between the two activities may be instrumental in maintaining glycolysis in pace with the distinct stage-specific requirements of carbohydrate metabolism.
刚地弓形虫两个发育阶段之间转换的精确分子机制，以及在此阶段转换过程中发生的代谢适应，目前尚不清楚。由于已知线粒体呼吸抑制剂会触发速殖子向缓殖子阶段的分化，我们认为一些转换成分可能存在于中心碳水化合物代谢的调节中。我们之前描述过一个编码缓殖子特异性烯醇化酶ENO1的cDNA。我们现在报告另一个编码烯醇化酶的cDNA（ENO2）的分离和特性，该cDNA在速殖子阶段优先表达。ENO1和ENO2推导的氨基酸序列具有73.65%的同一性。它们与植物烯醇化酶都有显著的同源性，存在两个类似植物的肽插入序列，一个五肽EWGW(Y)C(S)和一个二肽EK（或DK）。我们证明，单独缺失ENO1的五肽基序或与二肽一起缺失，会大幅降低对2PGA底物的亲和力，这表明陆地植物和顶复门寄生虫的烯醇化酶中这些肽的进化获得为其酶活性贡献了特定功能。刚地弓形虫ENO1和ENO2也在大肠杆菌中表达为活性重组酶。虽然ENO1和ENO2显示出相似的K(m)值，但与缓殖子特异性烯醇化酶（ENO1）相比，纯速殖子特异性酶（ENO2）在V(max)时的比活性高三倍。此外，使用针对重组酶产生的多克隆抗体进行的免疫印迹分析表明，速殖子和缓殖子中的天然烯醇化酶在抗原性上也不同。综上所述，我们的结果表明，两种活性之间的差异可能有助于使糖酵解与碳水化合物代谢的不同阶段特异性需求保持同步。
