CONSTRUCTION AND FUNCTION OF FUSION ENZYMES OF THE HUMAN CYTOCHROME-P450SCC SYSTEM

Type I cytochrome P450 enzyme systems are found in mitochondria and co nsist of three components, a flavoprotein (adrenodoxin reductase, AdRe d), an iron-sulfur protein (adrenodoxin, Adx), and the cytochrome P450 ; Type II P450 enzymes in the endoplasmic reticulum consist of only tw o components, P450 reductase and the P450. Genetically engineered fusi on proteins of Type II cytochromes P450 (such as steroid 17alpha- and 21-hydroxylases) produce enzymes with increased activity. To test the consequences of constructing fusions of Type I enzymes, we built fusio n proteins based on the cholesterol side-chain cleavage enzyme, P450sc c. We constructed expression vectors for three fusion proteins: NH2-P4 50scc-AdRed-COOH, P450-AdRed-Adx, and P450sec-Adx-AdRed. The various c omponents were assembled from cassette-like cDNA fragments modified an d amplified by polymerase chain reaction (PCR), subcloned into a speci ally tailored vector, and linked by DNA segments encoding hydrophilic linker peptides. The final vectors were transfected into COS-1 cells, incubated with 22R-hydroxycholesterol, and assayed by the secretion of pregnenolone into the culture medium. Triple transfection of three in dividual vectors expressing P450scc, AdRed, and Adx yielded more pregn enolone than did transfection with P450scc alone. The P450scc-AdRed an d P450scc-Adx-AdRed fusion proteins produced levels of pregnenolone si milar to the control triple transfection. However, the P450scc-AdRed-A dx fusion produced substantially more pregnenolone, having an apparent V(max) of 9.1 ng of pregnenolone produced per milliliter of medium pe r 24 hr, compared to a V(max) of 1.7 ng/ml per day for the triple tran sfection. These studies suggest that the association of the various co mponents of the cholesterol side-chain cleavage system is partly respo nsible for the slow conversion of cholesterol to pregnenolone in vivo, and that adrenodoxin is a key factor in determining the reaction rate . These studies also show that fusion proteins of Type I cytochrome P4 50 enzymes can be designed to increase enzymatic efficiency.