Schlösser Thomas, Gätgens Cornelia, Weber Ulrike, Stahmann K-Peter
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Yeast. 2004 Jan 15;21(1):63-73. doi: 10.1002/yea.1058.
Alanine : glyoxylate aminotransferase is one of three different enzymes used for glycine synthesis in Saccharomyces cerevisiae. The open reading frame YFL030w (named AGX1 in the following), encoding this enzyme, was identified by comparing enzyme specific activities in knockout strains. While 100% activity was detectable in the parental strain, 2% was found in a YFL030w::kanMX4 strain. The ORF found at that locus was suspected to encode alanine : glyoxylate aminotransferase because its predicted amino acid sequence showed 23% identity to the human homologue. Since the YFL030w::kanMX4 strain showed no glycine auxtrophic phenotype, AGX1 was replaced by KanMX4 in a Delta GLY1 Delta SHM1 Delta SHM2 background. These background mutations, which cause inactivation of threonine aldolase, mitochondrial and cytosolic serine hydroxymethyltransferase, respectively, lead to a conditional glycine auxotrophy. This means that growth is not possible on glucose but on ethanol as the sole carbon source. Additional disruption of AGX1 revealed a complete glycine auxotrophy. Complementation was observed by transformation with a plasmid-encoded AGX1.
乙醛酸氨基转移酶是酿酒酵母中用于甘氨酸合成的三种不同酶之一。通过比较敲除菌株中的酶比活性，鉴定出编码该酶的开放阅读框YFL030w（以下简称AGX1）。在亲本菌株中可检测到100%的活性，而在YFL030w::kanMX4菌株中仅发现2%的活性。在该位点发现的开放阅读框被怀疑编码丙氨酸：乙醛酸氨基转移酶，因为其预测的氨基酸序列与人类同源物有23%的同一性。由于YFL030w::kanMX4菌株没有甘氨酸营养缺陷型表型，在ΔGLY1ΔSHM1ΔSHM2背景下，AGX1被KanMX4取代。这些背景突变分别导致苏氨酸醛缩酶、线粒体和胞质丝氨酸羟甲基转移酶失活，导致条件性甘氨酸营养缺陷。这意味着在葡萄糖上无法生长，但在乙醇作为唯一碳源时可以生长。AGX1的进一步破坏显示出完全的甘氨酸营养缺陷。通过用质粒编码的AGX1转化观察到了互补作用。
