EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF A BILE ACID-INDUCIBLE 3-ALPHA-HYDROXYSTEROID DEHYDROGENASE FROM EUBACTERIUM SP STRAIN VPI-12708
EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF A BILE ACID-INDUCIBLE 3-ALPHA-HYDROXYSTEROID DEHYDROGENASE FROM EUBACTERIUM SP STRAIN VPI-12708
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DOI：
10.1007/bf00295498
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发表时间：
1995-05-01
影响因子：
2.6
通讯作者：
HYLEMON, PB
中科院分区：
文献类型：
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作者：
MALLONEE, DH;LIJEWSKI, MA;HYLEMON, PB
我们之前已经克隆并测序了来自真杆菌属胆汁酸诱导基因家族的三个成员。菌株VPI 12708，编码27,000-M(r)多肽。这些基因的两个拷贝（baiA1 和 baiA3）是相同的，而第三个拷贝（baiA2）编码与 baiA1 和 baiA3 基因产物具有 92% 氨基酸同一性的多肽。我们在大肠杆菌中过表达了baiA1基因并分析了表达活性。使用无细胞提取物对反应中的 C-14 标记胆汁酸产物进行薄层色谱分析，结果显示 BaiA1 蛋白具有 3 α-羟基类固醇脱氢酶活性。 BaiA1蛋白可以同时利用NAD(+)和NADP(+)，并且优选的类固醇底物是胆酰辅酶A缀合物而不是游离胆酸。这些结果表明 BaiA 蛋白是新型 3 α-羟基类固醇脱氢酶。
We have previously cloned and sequenced three members of a bile acid-inducible gene family from Eubacterium sp. strain VPI 12708 that encode 27,000-M(r) polypeptides. Two copies of these genes (baiA1 and baiA3) are identical, while the third copy (baiA2) encodes a polypeptide sharing 92% amino acid identity with the baiA1 and baiA3 gene products. We have overexpressed the baiA1 gene in Escherichia coli and analyzed the expressed activity. Thin-layer chromatography of C-14-labeled bile acid products from reactions using cell-free extracts revealed a 3 alpha-hydroxysteroid dehydrogenase activity for the BaiA1 protein. The BaiA1 protein could utilize both NAD(+) and NADP(+), and the preferred steroid substrate was the cholyl-coenzyme A conjugate rather than free cholic acid. These results show that the BaiA proteins are novel 3 alpha-hydroxysteroid dehydrogenases.