Drobnic K, Krizaj I, Gubensek F, Komel R
Institute of Biochemistry, Medical Faculty, Ljubljana, Slovenia.
Biochem Biophys Res Commun. 1993 Jan 29;190(2):509-15. doi: 10.1006/bbrc.1993.1077.
We have purified a steroid-inducible 1:2-dehydrogenase from Nocardia opaca. The final enzyme preparation was purified 120-fold with a recovery of 38%. The N-terminal amino acid sequence was determined to be: Met-Gln-Asp-Trp-Thr-Ser-Glu-(Cys)-Asp-Val-Leu-Val-Val-Gly-. From the genomic library of Nocardia opaca in the plasmid pUC19, a clone designated as pSTD23 containing a 0.9 kb KpnI-PstI fragment was found to hybridize with an oligonucleotide probe corresponding to the first six amino acids from the N-terminal of the purified protein. The nucleotide sequence of the upstream region and a part of the structural domain were determined. The sequence of the first 56 amino acids of the steroid 1:2-dehydrogenase from Nocardia opaca as deduced from its gene sequence showed a 58% homology with the corresponding gene from Pseudomonas testosteroni, and the conservative sequences in the FAD-binding domain were also determined.
我们从不透明诺卡氏菌中纯化出了一种类固醇诱导型1:2-脱氢酶。最终的酶制剂纯化了120倍，回收率为38%。测定其N端氨基酸序列为：甲硫氨酸-谷氨酰胺-天冬氨酸-色氨酸-苏氨酸-丝氨酸-谷氨酸-（半胱氨酸）-天冬氨酸-缬氨酸-亮氨酸-缬氨酸-缬氨酸-甘氨酸-。从质粒pUC19中的不透明诺卡氏菌基因组文库中，发现一个名为pSTD23的克隆含有一个0.9 kb的KpnI-PstI片段，它能与一个对应于纯化蛋白N端前六个氨基酸的寡核苷酸探针杂交。测定了上游区域和部分结构域的核苷酸序列。从不透明诺卡氏菌类固醇1:2-脱氢酶基因序列推导的最初56个氨基酸序列与睾丸酮假单胞菌的相应基因显示出58%的同源性，并且还确定了FAD结合结构域中的保守序列。
