Identification and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii
Identification and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii
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DOI：
10.1128/jb.179.16.5118-5125.1997
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发表时间：
1997-08-01
影响因子：
3.2
通讯作者：
Yamamoto, S
中科院分区：
文献类型：
--
作者：
Ikai, H;Yamamoto, S
CA。2.2-对鲍曼不动杆菌（Acinetobacter baumannii）L-2，4-二氨基丁酸脱羧酶（L-2，4-diaminobutyrate decarboxylase，DDC）基因上游150 bp的区域进行了序列测定，发现该基因含有另一个1，338个核苷酸的开放阅读框，编码一个分子量为47.323 Da的蛋白质，氨基酸序列同源性分析表明该基因产物属于氨基转移酶亚群II。当对大肠杆菌转化体的粗提物进行检查时，检测到催化以下可逆反应的新型氨基转移酶活性时，这一点首次得到证实：L-2，4-二氨基丁酸+ 7-酮基谷氨酸L-谷氨酸+L-天冬氨酸β-半醛。当该基因在E.大肠杆菌中表达，并将相应的蛋白纯化至均一。因此，将该基因及其产物分别命名为dar和L-2，4-二氨基丁酸：2-酮戊二酸4-氨基转移酶（DABA AT）。通过PCR扩增和蛋白质印迹法在其他不动杆菌中检测到dat和DABA AT的明显同源物。这些结果表明dat基因（以及dde基因）参与了1，3-丙二胺的合成，这是该属中发现的唯一的二胺。然而，生物作用，如果存在的话，1.3-丙二胺合成是未知的。
The ca. 2.2-kbp region upstream of the ddc gene encoding L-2,4-diaminobutyrate decarboxylase in Acinetobacter baumannii was sequenced and found to contain another open reading frame of 1,338 nucleotides encoding a protein with a deduced molecular mass of 47.323 Da. Analysis of the homologies observed from the deduced amino acid sequence indicated that the gene product is an enzyme belonging to subgroup II of the aminotransferases. This was first verified when examination of the crude extracts from Escherichia coli transformants led to detection of a novel aminotransferase activity catalyzing the following reversible reactions: L-2,4-diaminobutyric acid + 7-ketoglutaric acidL-glutamic acid + L-aspartic beta-semialdehyde. Further confirmation was obtained when the gene was overexpressed in E. coli and the corresponding protein was purified to homogeneity. It catalyzed the same reactions and its N-terminal amino acid sequence was consistent with that deduced from the nucleotide sequence, Therefore, the gene and its product were named dar and L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase (DABA AT), respectively, Feeding experiments of A. baumannii with L-[U-C-14]aspartic acid resulted in the incorporation of the label into 1,3-diaminopropane Apparent homologs of dat and DABA AT were detected in other Acinetobacter species by PCR amplification and Western blotting, These results indicate that the dat gene (as well as the dde gene) participates in the synthesis of 1,3-diaminopropane, the only diamine found in this genus. However, the biological role, if one exists, of 1.3-diaminopropane synthesis is unknown.