Construction and characterization of a catalytic fusion protein system

P-450(11β)-adrenodoxin reductase-adrenodoxin

Pei Rang Cao, Hannes E. Buelow, Bruno Dumas, Rita Bernhardt

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)253-264
Number of pages12
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1476
Issue number2
DOIs
StatePublished - Feb 9 2000
Externally publishedYes

Fingerprint

Adrenodoxin
Ferredoxin-NADP Reductase
Fusion reactions
Electrons
Proteins
Hydroxylation
Cortodoxone
COS Cells
Cytochrome P-450 Enzyme System
Hydrocortisone
Complementary DNA
Steroids
Peptides
Pharmaceutical Preparations

Keywords

  • Bovine P-450(11β)
  • Fusion protein construct
  • Human P-450(11β)
  • P- 450(11β)-adrenodoxin reductase-adrenodoxin
  • P-450(11β)-adrenodoxin- adrenodoxin reductase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Structural Biology
  • Biophysics

Cite this

Construction and characterization of a catalytic fusion protein system : P-450(11β)-adrenodoxin reductase-adrenodoxin. / Cao, Pei Rang; Buelow, Hannes E.; Dumas, Bruno; Bernhardt, Rita.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1476, No. 2, 09.02.2000, p. 253-264.

Research output: Contribution to journalArticle

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