Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism
Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism
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DOI：
10.1128/jb.186.22.7754-7762.2004
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发表时间：
2004-11-01
影响因子：
3.2
通讯作者：
van der Oost, J
中科院分区：
文献类型：
--
作者：
Ettema, TJG;Makarova, KS;van der Oost, J
尽管已经测量了磷酸烯醇丙酮酸羧化酶（PEPC）活性，并且在某些情况下甚至从某些古细菌中纯化，但尚未阐明负责该活性的基因。使用敏感的序列比较方法，我们检测到了一个高度保守的，未表征的古细胞基因家族，该家族与规范PEPC的催化核心密切相关。为了验证该古细胞基因家族的预测功能，我们从高疗酸性磺基磺胺solfataricus中克隆了一个代表，并在功能上产生了相应的酶，作为与大肠杆菌麦芽糖麦芽糖结合蛋白的融合。纯化的融合蛋白确实显示出高度可热的PEPC活性。 Solfataricus的特征性古细胞PEPC（ATPEPC）的结构和生化特性与先前报道的其他古细菌PEPC酶的生化分析非常吻合。新近识别的ATPEPC及其独特的特性构成了古细胞域中中央碳代谢途径酶多功能的另一个例子。
Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophille Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.