Michels P A, Marchand M, Kohl L, Allert S, Wierenga R K, Opperdoes F R
International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases, Brussels, Belgium.
Eur J Biochem. 1991 Jun 1;198(2):421-8. doi: 10.1111/j.1432-1033.1991.tb16031.x.
Trypanosoma brucei contains two isoenzymes for glyceraldehyde-3-phosphate dehydrogenase: one enzyme resides in a microbody-like organelle, the glycosome; the other is found in the cytosol. Previously we have reported the characterization of the gene for the glycosomal enzyme [Michels, P. A. M., Poliszczak, A., Osinga, K. A., Misset, O., Van Beeumen, J., Wierenga, R. K., Borst, P. & Opperdoes, F. R. (1986) EMBO J. 5, 1049-1056]. Here we describe the cloning and analysis of the gene that codes for the cytosolic isoenzyme. The gene encodes a polypeptide of 330 amino acids, with a calculated molecular mass of 35440 Da. The two isoenzymes are only 55% identical. The cytosolic glyceraldehyde-3-phosphate dehydrogenase differs from the glycosomal enzyme in the following respects: (a) its subunit molecular mass is 3.4 kDa smaller due to the absence of insertions and a small C-terminal extension which are unique to the glycosomal protein; (b) the cytosolic enzyme has a lower pI (7.9, as compared to 9.3 for the glycosomal isoenzyme), which is due to a reduction in the excess of positively charged amino acids (the calculated net charges of the polypeptides are +2 and +11, respectively). We have compared the amino acid sequences of the two T. brucei glyceraldehyde-3-phosphate dehydrogenases, with 24 available sequences of the corresponding enzyme of other organisms from various phylogenetic groups. On the basis of this comparison an evolutionary tree was constructed. This analysis strongly supports the theory that T. brucei diverged early in evolution from the main eukaryotic branch of the phylogenetic tree. Further, two separate branches for the lineages leading to Trypanosoma are inferred from the amino acid sequences, suggesting that the genes for the two glyceraldehyde-3-phosphate dehydrogenases of the trypanosome are distantly related and must have been acquired independently by the trypanosomal ancestor. The branching determined with the glycosomal enzyme precedes that found with the cytosolic enzyme. The available data do not allow us to decide which of the two genes originally belonged to the trypanosome lineage and which entered the cell later by horizontal gene transfer.
布氏锥虫含有两种3-磷酸甘油醛脱氢酶同工酶：一种酶存在于类似微体的细胞器——糖体中；另一种存在于细胞质中。此前我们已报道了糖体酶基因的特性[米歇尔，P.A.M.，波利什扎克，A.，奥辛加，K.A.，米塞特，O.，范·贝厄门，J.，维伦加，R.K.，博斯特，P.和奥珀多斯，F.R.（1986年）《欧洲分子生物学组织杂志》5，1049 - 1056]。在此我们描述编码细胞质同工酶的基因的克隆与分析。该基因编码一个由330个氨基酸组成的多肽，计算分子量为35440道尔顿。这两种同工酶仅有55%的同源性。细胞质3-磷酸甘油醛脱氢酶与糖体酶在以下方面存在差异：（a）由于缺少糖体蛋白特有的插入片段和小的C末端延伸，其亚基分子量小3.4千道尔顿；（b）细胞质酶的等电点较低（7.9，而糖体同工酶为9.3），这是由于带正电荷氨基酸过量的情况减少（多肽的计算净电荷分别为 +2和 +11）。我们已将两种布氏锥虫3-磷酸甘油醛脱氢酶的氨基酸序列与来自不同系统发育类群的其他生物的相应酶的24个可用序列进行了比较。基于此比较构建了进化树。该分析有力地支持了布氏锥虫在进化早期就从系统发育树的主要真核分支分化出来的理论。此外，从氨基酸序列推断出通向锥虫的谱系有两个独立分支，这表明锥虫的两种3-磷酸甘油醛脱氢酶基因关系较远，必定是由锥虫祖先独立获得的。由糖体酶确定的分支先于由细胞质酶确定的分支。现有数据无法让我们确定这两个基因中哪一个最初属于锥虫谱系，哪一个是后来通过水平基因转移进入细胞的。
