Sundström M, Lindqvist Y, Schneider G, Hellman U, Ronne H
Department Molecular Biology, Swedish University of Agricultural Science, Uppsala Biomedical Center.
J Biol Chem. 1993 Nov 15;268(32):24346-52.
A gene encoding transketolase, TKL1, was cloned from the budding yeast Saccharomyces cerevisiae using oligonucleotide primers derived from protein sequence data. The TKL1 sequence predicts a 74-kDa polypeptide which is related to other transketolases. A sequence comparison revealed that the transketolases can be subdivided into three evolutionary branches. We also found that the transketolases are related to another vitamin B1-dependent enzyme: the E1 subunit of pyruvate dehydrogenase from Escherichia coli. Gene disruption and overexpression experiments were used to investigate the function of transketolase in yeast. We found that growth on fermentable carbon sources, but not on gluconeogenic carbon sources, is reduced in cells disrupted for TKL1. This suggests that the glycolytic efficiency is impaired. Growth on fermentable carbon sources is also reduced in cells that overexpress TKL1. Finally, we found that cells disrupted for TKL1 are unable to grow in the absence of aromatic amino acids. This is most likely due to the fact that transketolase is required for the synthesis of erythrose-4-P, a precursor of the aromatic amino acids.
利用从蛋白质序列数据推导而来的寡核苷酸引物，从出芽酵母酿酒酵母中克隆出了一个编码转酮醇酶的基因TKL1。TKL1序列预测出一种与其他转酮醇酶相关的74 kDa多肽。序列比较显示，转酮醇酶可细分为三个进化分支。我们还发现，转酮醇酶与另一种维生素B1依赖酶有关：大肠杆菌丙酮酸脱氢酶的E1亚基。通过基因破坏和过表达实验来研究酵母中转酮醇酶的功能。我们发现，在TKL1基因被破坏的细胞中，利用可发酵碳源生长，但利用糖异生碳源生长不受影响，这表明糖酵解效率受损。在过表达TKL1的细胞中，利用可发酵碳源生长也会减少。最后，我们发现，TKL1基因被破坏的细胞在没有芳香族氨基酸的情况下无法生长。这很可能是因为转酮醇酶是芳香族氨基酸前体赤藓糖-4-磷酸合成所必需的。
