Construction and characterization of a catalytic fusion protein system: P-450(11beta)-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 procedures. The most critical step is the 11beta-hydroxylation of 11-deoxycortisol, which is catalyzed by a mitochondrial enzyme, P-450(11beta). Various fusion constructs of P-450(11beta) 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(11beta) 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(11beta)-adrenodoxin reductase-adrenodoxin was more active than that of P-450(11beta)-adrenodoxin-adrenodoxin reductase, 1.5- and 3-fold for bovine and human P-450(11beta), respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11beta)-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.

Modulation of aldosterone biosynthesis by adrenodoxin mutants with different electron transport efficiencies.

Aldosterone biosynthesis is highly regulated on different levels by hormones, potassium, lipid composition of the membrane and the molecular structure of its gene. Here, the influence of the electron transport efficiency from adrenodoxin (Adx) to CYP11B1 on the activities of bovine CYP11B1 has been investigated using a liposomal reconstitution system with truncated mutants of Adx. It could be clearly demonstrated that Adx mutants Adx 4-114 and Adx 4-108, possessing enhanced electron transfer abilities, produce increases in corticosterone and aldosterone biosynthesis. Based on the Vmax values of corticosterone and aldosterone formation, Adx 4-108 and Adx 4-114 enhance corticosterone synthesis 1.3-fold and aldosterone formation threefold and twofold, respectively. The production of 18-hydroxycorticosterone was changed only slightly in these Adx mutants. The effect of Adx 1-108 on the product patterns of bovine CYP11B1, human CYP11B1 and human CYP11B2 was confirmed in COS-1 cells by cotransfection of CYP11B- and Adx-containing expression vectors. It could be shown that Adx 1-108 enhances the formation of aldosterone by bovine CYP11B1 and by human CYP11B2, and stimulates the production of corticosterone by bovine CYP11B1 and human CYP11B1 and CYP11B2 also.

Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.