CYP17 is a steroidogenic enzyme located in the zona fasciculata and zona reticularis of the adrenal cortex and gonad tissues and which has dual functions -hydroxylation and as a lyase. The first activity gives hydroxylation of pregnenolone and progesterone at the C 17 position to generate 17 -hydroxypregnenolone and 17 -hydroxyprogesterone, while the second enzymic activity cleaves the C 17 -C 20 bond of 17 -hydroxypregnenolone and 17 -hydroxyprogesterone to form dehydroepiandrosterone and androstenedione respectively. The modulation of these two activities occurs through cytochrome b 5 . Association of cytochrome b 5 and CYP17 is thought to be based primarily on electrostatic interactions in which the negatively charged residues pair up with positively charged residues on the proximal surface of the CYP17 molecule. Non-specific interactions of the hydrophobic membrane regions of cytochrome b 5 and CYP17 are also thought to play a crucial role in the association of these two haemoproteins. Although cytochrome b 5 is known to stimulate CYP activity by contributing the second electron in the catalytic cycle, in the case of CYP17, the mechanism of cleavage stimulation proceeds via an allosteric mode. It is hypothesised that cytochrome b 5 promotes the cleavage by aligning the iron-oxygen complex attack onto the C 20 rather than the C 17 atom of the steroid substrate molecule. Thus, further understanding of the mechanism of modulation by cytochrome b 5 of the hydroxylase and lyase activities should shed new insights on developing therapeutic targets in CYP17-linked biochemical processes such as adrenarche, polycystic ovary syndrome and prostate cancer.

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b 5 /NADH cytochrome b 5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b 5 and NADH cytochrome b 5 reductase, we show here that fungal CYP51 mediated sterol 14K K-demethylation can be wholly and efficiently supported by the cytochrome b 5 /NADH cytochrome b 5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b 5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.z 1999 Federation of European Biochemical Societies.

Although there are a large number of protocols for the isolation of small quantities of plasmid DNA from bacterial cells (minipreps), this unit presents four procedures based on their speed and success: the alkaline lysis prep, a modification of the alkaline lysis prep that is performed in 1.5-ml tubes or 96-well microtiter dishes, the boiling method, and a lithium-based procedure. A support protocol provides information on storing plasmid DNA.

The soluble core domain of cytochrome b5 of liver endoplasmic reticulum was appended at its amino terminus to full-length alkaline phosphatase secretory signal sequence including the ribosomal binding site. The chimeric precursor gene was placed under the transcriptional control of the native pho promoter in a prokaryotic expression vector. Induction of Escherichia coli by growth in a phosphate-limited medium resulted in abundant synthesis of cytochrome b5 as detected spectrophotometrically and by visual transformation of the bacteria to a pink color. The signal-appended cytochrome b5, but not the corresponding signal-deficient derivative, was translocated across the bacterial inner membrane and processed to yield authentic, haem-assembled cytochrome b5 within the periplasm. The eventual processing of the chimeric cytochrome b5 precursor was unusual regarding the known reaction specificity of signal peptidase. The exported, mature haemoprotein was biochemically indistinguishable from its native mammalian counterpart. At peak induction, approximately 6 mg of correctly matured cytochrome b5 per liter of culture was exported. This amount of cytochrome b5 constituted 6% (w/w) of the periplasmic protein. The appearance of the exported apo-cytochrome b5 preceded the formation of holo-protein. Thus the eukaryotic cytoplasmic protein was efficiently exported from E. coli and post-translocationally modified to generate a functional haemoprotein in the periplasm.