Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450.
The steroid 11ß-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4-5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g  pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011ß system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011α from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11ß-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M(r) 60 kD) and a NADPH-cytochrome P450 reductase (M(r) 79 kD).
Max Delbrück Centre for Molecular Medicine, Berlin-Buch, Germany.
G R
GR
Jänig
D
D
Pfeil
M
M
Müller-Frohne
H
H
Riemer
M
M
Henning
W
W
Schwarze
K
K
Ruckpaul
D000935:Antifungal Agents / Q000494:pharmacology; D001203:Ascomycota / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D015153:Blotting, Western; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D025282:Mycelium / Q000201:enzymology / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D013237:Stereoisomerism; D013252:Steroid 11-beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D011374:Progesterone; D009249:NADP; D013252:Steroid 11-beta-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase