Improved purification of steroid 1:2-dehydrogenase from Nocardia opaca and partial characterization of its cloned gene sequence.
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.
Drobnic
K
K
Institute of Biochemistry, Medical Faculty, Ljubljana, Slovenia.
Krizaj
I
I
Gubensek
F
F
Komel
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009615:Nocardia / Q000201:enzymology* / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase