Basse C W, Lottspeich F, Steglich W, Kahmann R
Institut für Genetik und Mikrobiologie, Universität München, Germany.
Eur J Biochem. 1996 Dec 15;242(3):648-56. doi: 10.1111/j.1432-1033.1996.0648r.x.
The phytopathogenic basidiomycetc Ustilago maydis produces indole-3-acetic acid (IndCH2COOH) and indole-3-pyruvic acid (Ind-Prv) from tryptophan. Indole-3-acetaldehyde (IndCH2CH2O) is the common intermediate in the conversion of Ind-Prv and tryptamine to IndCH2COOH. We purified an enzyme (Iad1) from U. maydis that catalyzes the NAD(+)-dependent conversion of IndCH2CH2O to IndCH2COOH and isolated corresponding cDNA and genomic clones. The identity of the cDNA clone was confirmed by expression in Escherichia coli and demonstration of enzymatic activity. In U. maydis, iad1-null mutants were generated by gene replacement. The ability to convert IndCH2CH2O to IndCH2COOH was at least 100-fold reduced in U. maydis iad1-null mutants grown in medium with glucose as carbon source. However, the iad1-null mutants were not diminished in their capacity to produce IndCH2COOH from tryptophan, indicating that IndCH2COOH formation from tryptophan apparently proceeds in the absence of IndCH2CH2O dehydrogenase activity under these conditions. Iad1 expression was strongly induced during growth on ethanol while under these conditions iad1-null mutants were unable to grow. This reveals that iad1 is primarily engaged in the conversion of ethanol to acetate. In iad1-null mutants we detected an additional NAD(+)-dependent IndCH2CH2O dehydrogenase activity that was induced during growth on L-arabinose but repressed in the presence of D-glucose. In arabinose-containing medium the conversion of tryptophan to IndCH2COOH was approximately 5-fold reduced in wild-type strains but 10-15-fold reduced in iad1-null mutant strains compared to IndCH2COOH formation in glucose-containing medium. In addition, the formation of Ind-Prv from tryptophan was abolished in wild-type and iad1-null mutant strains. During growth on arabinose, the conversion of tryptamine to IndCH2COOH was strongly favored suggesting that the glucose-repressible IndCH2CH2O dehydrogenase is required to convert IndCH2CH2O derived from tryptamine to IndCH2COOH.
植物病原担子菌玉米黑粉菌可从色氨酸产生吲哚 - 3 - 乙酸（IndCH2COOH）和吲哚 - 3 - 丙酮酸（Ind - Prv）。吲哚 - 3 - 乙醛（IndCH2CH2O）是Ind - Prv和色胺转化为IndCH2COOH过程中的共同中间体。我们从玉米黑粉菌中纯化了一种酶（Iad1），它催化IndCH2CH2O依赖NAD(+)转化为IndCH2COOH，并分离出了相应的cDNA和基因组克隆。通过在大肠杆菌中的表达和酶活性的证明，证实了cDNA克隆的身份。在玉米黑粉菌中，通过基因替换产生了iad1基因缺失突变体。在以葡萄糖作为碳源的培养基中生长的玉米黑粉菌iad1基因缺失突变体，将IndCH2CH2O转化为IndCH2COOH的能力至少降低了100倍。然而，iad1基因缺失突变体从色氨酸产生IndCH2COOH的能力并未减弱，这表明在这些条件下，色氨酸形成IndCH2COOH显然是在没有IndCH2CH2O脱氢酶活性的情况下进行的。在乙醇上生长期间，Iad1表达被强烈诱导，而在这些条件下iad1基因缺失突变体无法生长。这表明iad1主要参与乙醇向乙酸的转化。在iad1基因缺失突变体中，我们检测到了另一种依赖NAD(+)的IndCH2CH2O脱氢酶活性，它在以L - 阿拉伯糖为碳源生长时被诱导，但在D - 葡萄糖存在时受到抑制。在含阿拉伯糖的培养基中，与在含葡萄糖的培养基中IndCH2COOH的形成相比，野生型菌株中色氨酸向IndCH2COOH的转化降低了约5倍，而iad1基因缺失突变体菌株中则降低了10 - 15倍。此外，野生型和iad1基因缺失突变体菌株中色氨酸向Ind - Prv的形成均被消除。在以阿拉伯糖为碳源生长期间，色胺向IndCH2COOH的转化受到强烈促进，这表明需要葡萄糖可抑制的IndCH2CH2O脱氢酶将来自色胺的IndCH2CH2O转化为IndCH2COOH。
