Construction and characterization of a catalytic fusion protein system: P-45011(beta)-adrenodoxin reductase-adrenodoxin

Cortisol is an important intermediate for the production of steroidal drugs and can only be synthesized chemically by rather complicated multi-step pr ocedures. The most critical step is the 11 beta-hydroxylation of 11-deoxyco rtisol, which is catalyzed by a mitochondrial enzyme, P-450(11 beta). Vario us fusion constructs of P-450(11 beta) with its electron transfer component s, adrenodoxin and adrenodoxin reductase, were produced by cDNA manipulatio n and were successfully expressed in COS-1 cells from which the hydroxylati on activities were assayed. It was demonstrated that the fusion protein req uired both adrenodoxin reductase and adrenodoxin for its activity and was n ot able to receive electrons from an external source. The fusion protein wi th all three components had less activity than P-450(11 beta) alone, receiv ing electrons from coexpressed or internal electron transfer components. Th e activities of the fusion proteins were determined mainly by the fusion se quence. The fusion protein with a sequence of P-450(11 beta)-adrenodoxin re ductase-adrenodoxin was more active than that of P-450(11 beta)-adrenodoxin -adrenodoxin reductase, 1.5- and 3-fold for bovine and human P-450(11 beta) , respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11 beta)-adre nodoxin reductase-adrenodoxin fusion protein indicated that the linker did not have significant effect on the P-450 activity. Taken together, the fusi on protein obtained here can serve as a model for the investigation of elec tron transfer in P-450 systems and is of potential importance for biotechno logical steroid production. (C) 2000 Elsevier Science B.V. All rights reser ved.