Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities.
Two soluble flavoproteins, purified from Escherichia coli cytosol and identified as flavodoxin and NADPH-flavodoxin (ferredoxin) reductase (flavodoxin reductase), have been found in combination to support the 17 alpha-hydroxylase activities of heterologously expressed bovine 17 alpha-hydroxylase cytochrome P450 (P450c17). Physical characteristics of the two flavoproteins including absorbance spectra, molecular weights, and amino-terminal sequences are identical with those reported previously for E. coli flavodoxin and flavodoxin reductase. Flavodoxin reductase, possessing FAD as a cofactor, is able to reconstitute P450c17 activities only in the presence of flavodoxin, an FMN-containing protein, and NAD(P)H. Reducing equivalents are utilized more effectively from NADPH than NADH by flavodoxin reductase. E. coli flavodoxin binds P450c17 directly and with relatively high affinity (apparent Ks approximately 0.2 microM) at low ionic strength, as evidenced by a change in spin state of the P450c17 heme iron upon titration with flavodoxin. This apparent spin shift is attenuated at moderate ionic strengths (100-200 mM KCl). In addition, bovine P450c17 binds reversibly to flavodoxin Sepharose in an ionic strength-dependent manner. These data implicate charge pairing as being important for the interaction between flavodoxin and P450c17. We propose that the amino acid sequence similarity between E. coli flavodoxin-flavodoxin reductase and the putative FMN, FAD, and NAD(P)H binding regions of cytochrome P450 reductase provides the basis for the reconstitution of P450c17 activities by this bacterial system.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
C M
CM
Jenkins
M R
MR
Waterman
D000446:Aldehyde-Lyases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013254:Steroid 17-alpha-Hydroxylase
D001426:Bacterial Proteins; D005418:Flavodoxin; D011994:Recombinant Proteins; D009243:NAD; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D000446:Aldehyde-Lyases