Expression of Catalytically Active Human Cytochrome P450scc inEscherichia coliand Mutagenesis of Isoleucine-462

Woods, Stephen T.; Sadleir, Jade; Downs, Tristan; Triantopoulos, Thrasivoulos; Headlam, Madeleine J.; Tuckey, Robert C.

doi:10.1006/abbi.1998.0621

Cited by 55 publications

(72 citation statements)

References 33 publications

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“…This scheme produced a large amount of partially pure enzyme with high activity (k cat ϭ 56 mol pregnenolone ⅐ min Ϫ1 ⅐ mol P450scc Ϫ1 with cholesterol as substrate) and was used for the large-scale synthesis of ergosterol derivatives. More highly purified P450scc was used for some small-scale incubations and was purified by phenyl Sepharose and DEAE-Sephacel (both from GE Healthcare, Rydalmere, NSW, Australia) chromatography as described previously (Woods et al, 1998). Both preparations gave similar results for ergosterol metabolism.…”

Section: Methodsmentioning

confidence: 97%

“…Human P450scc was expressed similarly to our previous report (Woods et al, 1998) but with pGro7 plasmid present (Tang et al, 2010), which increased the P450scc expression level from 35 nM to approximately 800 nM. The P450scc from a 1-liter culture was extracted from the bacterial membrane fraction as described previously (Woods et al, 1998) except that 1% sodium cholate (without Emulgen 911) was used. The extract was centrifuged at 107,000g for 60 min to remove insoluble debris, and the supernatant was applied to a 6 ϫ 2.5 cm hydroxyapatite column equilibrated with buffer comprising 20 mM potassium phosphate (pH 7.4), 0.1 mM dithiothreitol, 0.1 mM EDTA and 20% glycerol.…”

Section: Methodsmentioning

confidence: 99%

“…Human adrenodoxin and adrenodoxin reductase were expressed in Escherichia coli and were purified as described previously (Woods et al, 1998;Tuckey et al, 2011). Human P450scc was expressed similarly to our previous report (Woods et al, 1998) but with pGro7 plasmid present (Tang et al, 2010), which increased the P450scc expression level from 35 nM to approximately 800 nM. The P450scc from a 1-liter culture was extracted from the bacterial membrane fraction as described previously (Woods et al, 1998) except that 1% sodium cholate (without Emulgen 911) was used.…”

Section: Methodsmentioning

confidence: 99%

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Human Cytochrome P450scc (CYP11A1) Catalyzes Epoxide Formation with Ergosterol

Tuckey¹,

Nguyen²,

Chen³

et al. 2011

Drug Metab Dispos

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ABSTRACT:Cytochrome P450scc (P450scc) catalyzes the cleavage of the side chain of both cholesterol and the vitamin D 3 precursor, 7-dehydrocholesterol. The aim of this study was to test the ability of human P450scc to metabolize ergosterol, the vitamin D 2 precursor, and define the structure of the major products. P450scc incorporated into the bilayer of phospholipid vesicles converted ergosterol to two major and four minor products with a k cat of 53 mol ⅐ min

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Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Human Cytochrome P450scc (CYP11A1) Catalyzes Epoxide Formation with Ergosterol

Tuckey¹,

Nguyen²,

Chen³

et al. 2011

Drug Metab Dispos

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“…Mouse and human adrenodoxin and human adrenodoxin reductase were expressed in E. coli and purified as before (Woods et al, 1998;Tuckey and Sadleir, 1999;Tang et al, 2010bTang et al, , 2012. Mouse and human CYP27B1 were coexpressed in E. coli with the chaperonins, GroEL/ES, to facilitate correct protein folding, as described before (Tang et al, 2010b.…”

Section: Methodsmentioning

confidence: 99%

Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

et al. 2013

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CYP11A1 can hydroxylate vitamin D3 at carbons 17, 20, 22, and 23, producing a range of secosteroids which are biologically active with respect to their ability to inhibit proliferation and stimulate differentiation of various cell types, including cancer cells. As 1a-hydroxylation of the primary metabolite of CYP11A1 action, 20S-hydroxyvitamin D3 [20(OH)D3], greatly influences its properties, we examined the ability of both human and mouse CYP27B1 to 1a-hydroxylate six secosteroids generated by CYP11A1. Based on their k cat /K m values, all CYP11A1-derived metabolites are poor substrates for CYP27B1 from both species compared with 25-hydroxyvitamin D3. No hydroxylation of metabolites with a 17a-hydroxyl group was observed. 17a,20-Dihydroxyvitamin D3 acted as an inhibitor on human CYP27B1 but not the mouse enzyme. We also tested CYP27B1 activity on 20,24-, 20,25-, and 20,26-dihydroxyvitamin D3, which are products of CYP24A1 or CYP27A1 activity on 20(OH)D3. All three compounds were metabolized with higher catalytic efficiency (k cat /K m ) by both mouse and human CYP27B1 than 25-hydroxyvitamin D3. CYP27B1 action on these new dihydroxy derivatives was confirmed to be 1a-hydroxylation by mass spectrometry and nuclear magnetic resonance analyses. Both 1,20,25-and 1,20,26-trihydroxyvitamin D3 were tested for their ability to inhibit melanoma (SKMEL-188) colony formation, and were significantly more active than 20(OH)D3. This study shows that CYP11A1-derived secosteroids are 1a-hydroxylated by both human and mouse CYP27B1 with low catalytic efficiency, and that the presence of a 17a-hydroxyl group completely blocks 1a-hydroxylation. In contrast, the secondary metabolites produced by subsequent hydroxylation of 20(OH)D3 at C24, C25, or C26 are very good substrates for CYP27B1.

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“…The concentration of cytochrome P450scc was determined from the CO-reduced minus reduced difference spectrum using an extinction coefficient of 91,000 M −1 cm −1 for the absorbance difference between 450 nm and 490 nm (Omura and Sato, 1964). Adrenodoxin was expressed in Escherichia coli and purified as previously described (Woods et al, 1998). N-62 StAR was a gift from Walter Miller (University of California, San Francisco).…”

Section: Preparation Of Enzymes and Hydroxyvitamin D3 Derivativesmentioning

confidence: 99%

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

Tuckey

Nguyen

Slominski

2008

The International Journal of Biochemistry & Cell Biology

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Vitamin D3 can be hydroxylated sequentially by cytochrome P450scc (CYP11A1) producing 20-hydroxyvitamin D3, 20,23-dihydroxyvitamin D3 and 17,20,23-trihydroxyvitamin D3. The aim of this study was to characterize the ability of vitamin D3 to associate with phospholipid vesicles and to determine the kinetics of metabolism of vitamin D3 by P450scc in vesicles and in 2-hydroxypropyl-β-cyclodextrin (cyclodextrin). Gel filtration of phospholipid vesicles showed that the vitamin D3 remained quantitatively associated with the phospholipid membrane. Vitamin D3 exchanged between vesicles at a rate 3.8-fold higher than for cholesterol exchange and was stimulated by N-62 StAR protein. The K m of P450scc for vitamin D3 in vesicles was 3.3 mol vitamin D3/mol phospholipid and the rate of conversion of vitamin D3 to 20-hydroxyvitamin D3 was first order with respect to the vitamin D3 concentration for the range of concentrations of vitamin D3 that could be incorporated into the vesicle membrane. 20-Hydroxyvitamin D3 was further hydroxylated by P450scc in vesicles, producing primarily 20,23-dihydroxyvitamin D3, with K m and k cat values 22-and 6-fold lower than those for vitamin D3, respectively. 20,23-Dihydroxyvitamin D3 was converted to 17,20,23-trihydroxyvitamin D3 with even lower K m and k cat values. Vitamin D3 and cholesterol were metabolized with comparable efficiencies in cyclodextrin, but the K m for both showed a strong dependence on the cyclodextrin concentration, decreasing with decreasing cyclodextrin. This study shows that vitamin D3 quantitatively associates with phospholipid vesicles, can exchange between membranes, and can be hydroxylated by membrane-associated P450scc but with lower efficiency than for cholesterol hydroxylation. The k cat values for metabolism of vitamin D3 in vesicles and 0.45% cyclodextrin are similar, but the ability to solubilize vitamin D3 at a concentration higher than its K m makes the cyclodextrin system more efficient for producing the hydroxyvitamin D3 metabolites for further characterization.

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Expression of Catalytically Active Human Cytochrome P450scc inEscherichia coliand Mutagenesis of Isoleucine-462

Cited by 55 publications

References 33 publications

Human Cytochrome P450scc (CYP11A1) Catalyzes Epoxide Formation with Ergosterol

Human Cytochrome P450scc (CYP11A1) Catalyzes Epoxide Formation with Ergosterol

Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

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