Purification and characterization of the 3-ketosteroid-delta 1-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans.
The 3-ketosteroid-delta 1-dehydrogenase (KS1DH) gene of Arthrobacter simplex IFO12069 cloned in Streptomyces lividans was overexpressed, resulting in production of the enzyme both extracellularly and intracellularly. The enzyme was purified by ammonium sulfate fractionation and chromatographies using DEAE-Toyopearl, Butyl-Toyopearl and Toyopearl HW55S from the supernatant of culture broth and cell-free extracts of S. lividans, and both preparations showed the same characteristics. The N-terminal amino acid sequence of both KS1DHs was M-D-W-A-E-E-Y-D, which coincided with the amino acid sequence deduced from the nucleotide sequence. Thus, the extracellular enzyme may derived from leakage of S. lividans cells during cultivation rather than secretion by processing of the signal sequence. The molecular weight of the enzyme was about 55,000, identical with the size deduced from the nucleotide sequence (M(r) 54,329). The optimum conditions for its activity were pH 10.0 and 40 degrees C. The enzyme catalyzed the conversion of several 3-keto-steroids, but those containing 11 alpha- or 11 beta-hydroxyl group were converted at low rates. The amino acid sequence of KS1DH from A. simplex is similar to those of KS1DH of Pseudomonas testosteroni and fumarate reductase from Shewanella putrefaciens.
Choi
K P
KP
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University.
Molnár
I
I
Yamashita
M
M
Murooka
Y
Y
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D005109:Extracellular Matrix / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D013256:Steroids; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase