Yurimoto H, Hasegawa T, Sakai Y, Kato N
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.
Yeast. 2000 Sep 30;16(13):1217-27. doi: 10.1002/1097-0061(20000930)16:13<1217::AID-YEA616>3.0.CO;2-2.
A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii. The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C. boidinii. We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes.
一种甲基营养酵母，博伊丁假丝酵母（Candida boidinii），在以D-丙氨酸作为唯一碳源或氮源生长时表现出D-氨基酸氧化酶活性（DAO，EC 1.4.3.3）。编码DAO的结构基因（DAO1）从博伊丁假丝酵母的基因组文库中克隆得到。该1035碱基对的基因编码345个氨基酸，预测的氨基酸序列与来自其他生物体的DAO具有显著相似性。通过一步基因敲除在博伊丁假丝酵母基因组中破坏了DAO1基因。缺失DAO1的菌株不能以D-丙氨酸作为碳源生长，但能以D-丙氨酸作为唯一氮源（以葡萄糖作为碳源）生长。这些结果表明，虽然DAO在以D-丙氨酸作为碳源的生长过程中起关键作用，但在博伊丁假丝酵母中应该存在另一种将D-丙氨酸作为氮源进行代谢的酶系统。我们还表明，DAO的三个C末端氨基酸序列-AKL对于DAO导入过氧化物酶体是必要且充分的。
