Molecular cloning and heterologous expression of progesterone 5beta-reductase from Digitalis lanata Ehrh.
A full-length cDNA clone that encodes progesterone 5beta-reductase (5beta-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5beta-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The K(m) and v(max) values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg(-1) protein, respectively. Only 5beta-pregnane-3,20-dione but not its alpha-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5beta-POR gene in D. lanata was determined by Southern blot analysis. The 5beta-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species.
Herl
Vanessa
V
Lehrstuhl für Pharmazeutische Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany. vherl@biologie.uni-erlangen.de
Fischer
Gabriele
G
Müller-Uri
Frieder
F
Kreis
Wolfgang
W
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002298:Cardenolides / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D004070:Digitalis / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013256:Steroids / Q000378:metabolism; D013379:Substrate Specificity
D002298:Cardenolides; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013256:Steroids; D009249:NADP; D010088:Oxidoreductases; C090493:progesterone 5 beta-reductase