Construction and Function of Fusion Enzymes of the Human Cytochrome P450scc System

Jennifer A. Harikrishna, Stephen M. Black, Grazyna D. Szklarz, Walter L. Miller

Research output: Contribution to journalArticlepeer-review

136 Scopus citations


Type I cytochrome P450 enzyme systems are found in mitochondria and consist of three components, a flavoprotein (adrenodoxin reductase, AdRed), an iron-sulfur protein (adrenodoxin, Adx), and the cytochrome P450; Type II P450 enzymes in the endoplasmic reticulum consist of only two components, P450 reductase and the P450. Genetically engineered fusion proteins of Type II cytochromes P450 (such as steroid 17α- and 21-hydroxylases) produce enzymes with increased activity. To test the consequences of constructing fusions of Type I enzymes, we built fusion proteins based on the cholesterol side-chain cleavage enzyme, P450scc. We constructed expression vectors for three fusion proteins: NH2-P450scc-AdRed-COOH, P450-AdRed-Adx, and P450scc-Adx-AdRed. The various components were assembled from cassette-like cDNA fragments modified and amplified by polymerase chain reaction (PCR), subcloned into a specially 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 individual vectors expressing P450scc, AdRed, and Adx yielded more pregnenolone than did transfection with P450scc alone. The P450scc-AdRed and P450scc-Adx-AdRed fusion proteins produced levels of pregnenolone similar to the control triple transfection. However, the P450scc-AdRed-Adx fusion produced substantially more pregnenolone, having an apparent Vmax of 9.1 ng of pregnenolone produced per milliliter of medium per 24 hr, compared to a Vmax of 1.7 ng/ml per day for the triple transfection. These studies suggest that the association of the various components of the cholesterol side-chain cleavage system is partly responsible 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 P450 enzymes can be designed to increase enzymatic efficiency.

Original languageEnglish (US)
Pages (from-to)371-379
Number of pages9
JournalDNA and cell biology
Issue number5
StatePublished - Jun 1993
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology


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