Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli.
The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.
Zöllner
Andy
A
Department of Biochemistry, Saarland University, Saarbrücken, Germany.
Kagawa
Norio
N
Waterman
Michael R
MR
Nonaka
Yasuki
Y
Takio
Koji
K
Shiro
Yoshitsugu
Y
Hannemann
Frank
F
Bernhardt
Rita
R
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019405:Cytochrome P-450 CYP11B2 / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005288:Ferredoxins / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
C081714:2Fe-2S ferredoxin; D005288:Ferredoxins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase