TY - JOUR
T1 - Construction and characterization of a catalytic fusion protein system
T2 - P-450(11β)-adrenodoxin reductase-adrenodoxin
AU - Cao, Pei Rang
AU - Bülow, Hannes
AU - Dumas, Bruno
AU - Bernhardt, Rita
N1 - Funding Information:
This work was supported in part by the Marie Curie fellowship from the European Commission and by a grant of the Stiftungsfonds Schering AG im Stifterverband für die Deutsche Wissenschaft to P.R.C. The authors, in addition, appreciate the support from the Deutsche Forschungsgemeinschaft (Be1343/2–5) and the Fonds der Chemischen Industrie. We thank Mrs. Susan Lloyd MacGilp (University of Edinburgh) for critical reading of the manuscript and improving the English language.
PY - 2000/2/9
Y1 - 2000/2/9
N2 - Cortisol is an important intermediate for the production of steroidal drugs and can only be synthesized chemically by rather complicated multi-step procedures. The most critical step is the 11β-hydroxylation of 11- deoxycortisol, which is catalyzed by a mitochondrial enzyme, P-450(11β). Various fusion constructs of P-450(11β) with its electron transfer components, adrenodoxin and adrenodoxin reductase, were produced by cDNA manipulation and were successfully expressed in COS-1 cells from which the hydroxylation activities were assayed. It was demonstrated that the fusion protein required both adrenodoxin reductase and adrenodoxin for its activity and was not able to receive electrons from an external source. The fusion protein with all three components had less activity than P-450(11β) alone, receiving electrons from coexpressed or internal electron transfer components. The activities of the fusion proteins were determined mainly by the fusion sequence. The fusion protein with a sequence of P-450(11β)- adrenodoxin reductase-adrenodoxin was more active than that of P-450(11β)- adrenodoxin-adrenodoxin reductase, 1.5- and 3-fold for bovine and human P- 450(11β), respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11β)- adrenodoxin reductase-adrenodoxin fusion protein indicated that the linker did not have significant effect on the P-450 activity. Taken together, the fusion protein obtained here can serve as a model for the investigation of electron transfer in P-450 systems and is of potential importance for biotechnological steroid production. (C) 2000 Elsevier Science B.V.
AB - Cortisol is an important intermediate for the production of steroidal drugs and can only be synthesized chemically by rather complicated multi-step procedures. The most critical step is the 11β-hydroxylation of 11- deoxycortisol, which is catalyzed by a mitochondrial enzyme, P-450(11β). Various fusion constructs of P-450(11β) with its electron transfer components, adrenodoxin and adrenodoxin reductase, were produced by cDNA manipulation and were successfully expressed in COS-1 cells from which the hydroxylation activities were assayed. It was demonstrated that the fusion protein required both adrenodoxin reductase and adrenodoxin for its activity and was not able to receive electrons from an external source. The fusion protein with all three components had less activity than P-450(11β) alone, receiving electrons from coexpressed or internal electron transfer components. The activities of the fusion proteins were determined mainly by the fusion sequence. The fusion protein with a sequence of P-450(11β)- adrenodoxin reductase-adrenodoxin was more active than that of P-450(11β)- adrenodoxin-adrenodoxin reductase, 1.5- and 3-fold for bovine and human P- 450(11β), respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11β)- adrenodoxin reductase-adrenodoxin fusion protein indicated that the linker did not have significant effect on the P-450 activity. Taken together, the fusion protein obtained here can serve as a model for the investigation of electron transfer in P-450 systems and is of potential importance for biotechnological steroid production. (C) 2000 Elsevier Science B.V.
KW - Bovine P-450(11β)
KW - Fusion protein construct
KW - Human P-450(11β)
KW - P- 450(11β)-adrenodoxin reductase-adrenodoxin
KW - P-450(11β)-adrenodoxin- adrenodoxin reductase
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U2 - 10.1016/S0167-4838(99)00243-5
DO - 10.1016/S0167-4838(99)00243-5
M3 - Article
C2 - 10669790
AN - SCOPUS:0343920070
SN - 0167-4838
VL - 1476
SP - 253
EP - 264
JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
IS - 2
ER -