Kirimura Kohtaro, Yoda Masashi, Kumatani Masaki, Ishii Yoshitaka, Kino Kuniki, Usami Shoji
Department of Applied Chemistry, School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan.
J Biosci Bioeng. 2002;93(2):136-44. doi: 10.1263/jbb.93.136.
The complementary DNA (cDNA) and chromosomal DNA (icdA) encoding the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Two cDNA clones (cDNA-1, 2.0 kb; cDNA-2, 1.5 kb) were obtained and sequenced, and an ORF of 1494 base pairs (bp) encoding a protein of 498 amino acids (aa) was identified in cDNA-1. The predicted amino acid sequence showed 73% and 67% sequence identities with those of the mitochondrial NADP+-ICDHs from Saccharomyces cerevisiae and pig, respectively. The sequence analysis of cDNA-1 and -2 revealed that the cDNA-2 lacks a 500-bp fragment from cDNA-1 which contains a mitochondrial targeting motif. A peroxisomal targeting motif at the C-terminus was found on the aa sequences of cDNA-1 and cDNA-2, but the cDNA-2 product seemed to be localized in the cytoplasm since the peroxisomes were not found in the mycelia of WU-2223L cultivated under the conditions of citric acid production. The expression of both cDNAs in Escherichia coli DEK2004, an isocitrate dehydrogenase-deficient mutant, revealed that both cDNAs complemented the glutamate-requiring phenotype, and that the transformants retained NADP(+)-ICDH activities. Therefore, it was clarified that both of the cDNA-1 and -2 products are fully functional. The chromosomal DNA, icdA, was cloned to correspond to cDNA-1, and its nucleotide sequence revealed that it contains seven introns. Southern hybridization using cDNA-1 and cDNA-2 indicated that there is only one copy of icdA on the chromosomes of A. niger WU-2223L. Northern hybridization analysis as for total RNA of WU-2223L revealed that two mRNAs of different sizes, 2.0 kb and 1.5 kb, were hybridized to the ORF of cDNA-1 used as a probe. Therefore, it was found that approximately 1500-nt and 2000-nt mRNAs were transcribed from only one icdA chromosomal gene in A. niger. Such a transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.
克隆了产柠檬酸的黑曲霉WU - 2223L编码NADP⁺特异性异柠檬酸脱氢酶（EC 1.1.1.42）的互补DNA（cDNA）和染色体DNA（icdA）。获得了两个cDNA克隆（cDNA - 1，2.0 kb；cDNA - 2，1.5 kb）并进行了测序，在cDNA - 1中鉴定出一个1494个碱基对（bp）的开放阅读框（ORF），其编码一个498个氨基酸（aa）的蛋白质。预测的氨基酸序列与酿酒酵母和猪的线粒体NADP⁺ - ICDHs的氨基酸序列分别具有73%和67%的序列同一性。对cDNA - 1和 - 2的序列分析表明，cDNA - 2缺少来自cDNA - 1的一个500 - bp片段，该片段包含一个线粒体靶向基序。在cDNA - 1和cDNA - 2的氨基酸序列上发现了C末端的过氧化物酶体靶向基序，但由于在柠檬酸生产条件下培养的WU - 2223L菌丝体中未发现过氧化物酶体，cDNA - 2产物似乎定位于细胞质中。这两个cDNA在异柠檬酸脱氢酶缺陷型突变体大肠杆菌DEK2004中的表达表明，这两个cDNA都能互补需要谷氨酸的表型，并且转化体保留了NADP(+) - ICDH活性。因此，明确了cDNA - 1和 - 2的产物都具有完全功能。克隆了与cDNA - 1对应的染色体DNA icdA，其核苷酸序列显示它含有7个内含子。使用cDNA - 1和 - 2进行的Southern杂交表明，黑曲霉WU - 2223L的染色体上icdA只有一个拷贝。对WU - 2223L总RNA的Northern杂交分析表明，两种不同大小的mRNA，2.0 kb和1.5 kb，与用作探针的cDNA - 1的ORF杂交。因此，发现黑曲霉中仅从一个icdA染色体基因转录出约1500 - nt和2000 - nt的mRNA。在任何其他生物体中，尚未观察到作为三羧酸循环关键调节酶之一的ICDH有这样的转录情况。
