The University of Western Australia

UWA Staff Profile

Ipsum Lorem

Rob Tuckey

Assoc/Prof Robert Tuckey

Associate Professor
Molecular Sciences, School of

Contact details
Address
School of Molecular Sciences
The University of Western Australia (M310)
35 Stirling Highway
CRAWLEY WA 6009
Australia
Phone
+61 8 6488 3040
Fax
+61 8 6488 1148
Email
robert.tuckey@uwa.edu.au
Location
Room 371, Bayliss Building, Perth campus
Qualifications
BSc PhD W.Aust.
Key research
Current research involves structure-function studies on cytochrome P450scc, regulation of progesterone synthesis in the human placenta, cholesterol transport for steroid synthesis, and metabolism of vitamins D2 and D3 and their precursors by cytochrome P450scc.
Publications
1.Slominski, A., Semak, I., Wortsman, J., Zjawiony, J., Li, W., Zbytek, B. and Tuckey, R.C. An alternate pathway of vitamin D2 metabolism: Cytochrome P450scc (CYP11A1)-mediated conversion to 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2, The FEBS Journal, in press (2006)
2.Slominski, A., Semak, I., Zjawiony, J., Wortsman, J., Gandy, M.N., Li, J., Zbytek, B., Li, W. and Tuckey, R.C. Enzymatic Metabolism of Ergosterol by Cytochrome P450scc to Biologically Active 17α, 24-Dihydroxyergosterol, Chemistry & Biology, 12: pp 931-939 (2005)
3.Slominski, A., Semak, I., Zjawiony, J., Wortsman, J., Wei, L., Szczesniewski, A. and Tuckey, R.C. The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism, The FEBS Journal, 272 : pp 4080-4090 (2005)
4.Tuckey, R.C. Progesterone Synthesis by the Human Placenta, Placenta, 26: pp 273-281 (Review) (2005)
5.Slominski, A., Zjawiony, J., Wortsman, J., Semak, I., Stewart, J., Pisarchik, A., Sweatman, T., Marcos, J., Dunbar, C. and Tuckey, R.C. A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin, European Journal of Biochemistry, 271: pp 4178-4188 (2004)
6.Tuckey, R.C., Bose, H., Czerwionka, I. and Miller, W.L. Molten Globule Structure and Steroidogenic Activity of N-218 MLN64 in Human Placental Mitochondria, Endocrinology, 145:4, pp 1700-1707 (2004)
7.Johnson, D., Norman, S., Tuckey, R.C. and Martin, L.L. Electrochemical behaviour of human adrenodoxin on a pyrolytic graphite electrode, Bioelectrochemistry, 59: pp 41-47 (2003)
8.Headlam, M.J., Wilce, M.C. and Tuckey, R.C. The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane, Biochimica et Biophysica Acta, 1617: pp 96-108 (2003)
9.Tuckey, R.C., Headlam, M.J., Bose, H.S. and Miller, W.L. Transfer of Cholesterol between Phospholipid Vesicles Mediated by the Steroidogenic Acute Regulatory Protein (StAR), The Journal of Biological Chemistry, 277:49, December 06, pp 47123-47128 (2002)
10.Tuckey, R.C. and Headlam, M.J. Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase, Journal of Steroid Biochemistry and Molecular Biology, 81: pp 153-158 (2002)
11.Headlam, M.J. and Tuckey, R.C. The effect of glycerol on cytochrome P450scc (CYP11A1) spin state, activity, and hydration, Archives of Biochemistry and Biophysics, 407: pp 95-102 (2002)
12.Tuckey, R.C., McKinley, A.J. and Headlam, M.J. Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta, European Journal of Biochemistry, 268: pp 2338-2343 (2001)
13.Tuckey, R. C. McKinley, A. and Sadleir, J. "Adrenodoxin Reductase Limits Cytochrome P450scc Activity in the Human Placenta" in Molecular Steroidogenesis, (Okamoto, M., Ishimura, Y. and Nawata, H. eds) pp 119-120 (2000). Universal Academy Press, Tokyo.
14.Tuckey, R. C. and Sadleir, J. The Concentration of Adrenodoxin Reductase Limits Cytochrome P450scc Activity in the Human Placenta. Eur. J. Biochem. 263: pp 319-325 (1999)
15.Kisselev, P., Tuckey, R. C., Woods, S., Triantopolous, T. and Schwarz, D. Enzymatic properties of Vesicle-Reconstituted Human Cytochrome P450scc (CYP11A1). Differences in Functioning of the Mitochondrial Electron Transfer Chain using Human and Bovine Adrenodoxin and Activation by Cardiolipin. Eur. J. Biochem. 260: pp 768-773 (1999)
16.Woods, S. T., Sadleir, J., Downs, T., Triantopoulos, T., Headlam, M. J. and Tuckey, R. C. Expression of Catalytically Active Human Cytochrome P450scc in Escherichia coli and Mutagenesis of Isoleucine-462. Arch. Biochem. Biophys. 353: pp 109-115 (1998)
17.Glencross, B., Mullan, B. P., Tuckey, R. C. and Hartmann, P. E. A Simplification of the Deuterium Oxide Dilution Technique using FT-IR Analysis of Plasma, for Estimating Piglet Milk Intake. Aust. J. Agric. Res. 48: pp 1099-1104 (1997)
18.Tuckey, R. C., Woods, S. T. and Tajbakhsh, M. Electron Transfer to Cytochrome P-450scc Limits Cholesterol Side-Chain-Cleavage Activity in the Human Placenta. Eur. J. Biochem. 244: pp 835-839 (1997)
19.Tuckey, R. C., Lawrence, J. and Cameron, K. J. Side Chain Cleavage of Cholesterol Esters by Human Cytochrome P-450scc. J. Steroid. Biochem. Mol. Biol. 58: pp 605-610 (1996)
20.Jennings, G. J., Sechi, S., Stevenson, P. M., Tuckey, R. C., Parmellee, D. and Mcalister-Henn, L. Cytoplasmic NADP+-Dependent Isocitrare Dehydrogenase: Isolation of Rat cDNA and Study of the Tissue Specific and Developmental Expression of mRNA. J. Biol. Chem. 269: pp 23128-23134 (1994)
21.Tuckey, R. C., Kostadinovic, Z. and Cameron, K. J. Cytochrome P-450scc Activity and Substrate Supply in Human Placental Trophoblasts. Mol. Cell Endocr. 105: pp 103-109 (1994)
22.Tuckey, R.C. and Cameron, K.J. Side Chain Specificities of Human and Bovine Cytochromes P-450scc. Eur. J. Biochem. 217: pp 209-215 (1993)
23.Tuckey, R.C. and Cameron, K.J. Catalytic properties of Cytochrome P-450scc Purified from the Human Placenta: Comparison to Bovine Cytochrome P-450scc. Biochim. Biophys. Acta 1163: pp 185-194 (1993)
24.Tuckey, R.C. and Cameron, K.J. Human Placental Cholesterol Side-Chain Cleavage: Enzymatic Synthesis of (22R)-20a,22-Dihydroxycholesterol. Steroids 58: pp 230-233 (1993)
25.Tuckey, R.C. Cholesterol Side Chain Cleavage by Mitochondria from the Human Placenta : Studies using Hydroxycholesterols as Substrates. J. Steroid Biochem. Molec. Biol. 42: pp 883-890 (1992)
26.Tuckey, R.C. Side Chain Cleavage of Cholesterol Sulfate by Ovarian Mitochondria. J. Steroid Biochem. Molec. Biol. 37: pp 121-127 (1990)
27.Tuckey, R.C. and Atkinson, H. Pregnenolone Synthesis from Cholesterol and Hydroxycholesterols by Mitochondria from Ovaries Following the Stimulation of Immature Rats with Pregnant Mare's Serum Gonadotropin and Human Choriogonadotropin. Eur. J. Biochem. 186: pp 255-259 (1989)
28.Tuckey, R.C. and Holland, J.W. Comparison of Pregnenolone Synthesis by Cytochrome P-450scc in Mitochondria from Porcine Corpora Lutea and Granulosa Cells of Follicles. J. Biol. Chem. 264: pp 5704-5709 (1989)
29.Tuckey, R.C., Kostadinovic, Z. and Stevenson, P.M. Ferredoxin and Cytochrome P-450scc Concentrations in Granulosa Cells of Porcine Ovaries during Follicular Cell Growth and Luteinization. J. Steroid Biochem. 31: pp 201-205 (1988)
30.Tuckey, R.C. and Stevenson, P.M. Ferredoxin Reductase Levels in the Ovaries of Pigs and Superovulated Rats during Follicular Cell Growth and Luteinization. Eur. J. Biochem. 161: pp 629-633 (1986)
31.O'Meara, M.L., Tuckey, R.C. and Stevenson, P.M. A Comparison of the Lipid Classes and Essential Fatty Acid Content of Rat Plasma Lipoproteins and Ovary. Int. J. Biochem. 17: pp 1027-1030 (1985)
32.Tuckey, R.C. and Stevenson, P.M. Purification and Analysis of Phospholipids in the Inner Mitochondrial Membrane Fraction of the Bovine Corpus Luteum, and Properties of Cytochrome P-450scc Incorporated into Vesicles Prepared from these Phospholipids. Eur. J. Biochem. 148: pp 379-384 (1985)
33.Tuckey, R.C., Lee, G., Costa, N.D. and Stevenson, P.M. The Composition and Distribution of Lipid Granules in the Rat Ovary. Molec. Cell. Endocr. 38: pp 187-195 (1984)
34.Tuckey, R.C. and Stevenson, P.M. Properties of Bovine Luteal Cytochrome P-450scc Incorporated into Artificial Phospholipid Vesicles. Int. J. Biochem. 16: pp 497-503 (1984)
35.Tuckey, R.C. and Stevenson, P.M. Properties of Ferredoxin Reductase and Ferredoxin from the Bovine Corpus Luteum. Int. J. Biochem. 16: pp 489-495 (1984)
36.Tuckey, R.C. and Kamin, H. Kinetics of O2 and CO Binding to Adrenal Cytochrome P-450scc: Effect of Cholesterol, Intermediates and Phosphatidylcholine Vesicles. J. Biol. Chem. 258: pp 4232-4237 (1983)
37.Tuckey, R.C. and Kamin, H. The Oxyferro Complex of Adrenal Cytochrome P-450scc: Effect of Cholesterol and Intermediates on its Stability and Optical Characteristics. J. Biol. Chem. 257: pp 9309-9314 (1982)
38.Lambeth, J.D., Kitchen, S.F., Farooqui, A.A., Tuckey, R.C. and Kamin, H. Cytochrome P-450scc - Substrate Interactions: Studies of Binding and Catalytic activity using Hydroxy-Cholesterols. J. Biol. Chem. 257: pp 1876-1884 (1982)
39.Tuckey, R.C. and Kamin, H. Kinetics of the Incorporation of Adrenal Cytochrome P-450scc into Phosphatidylcholine Vesicles. J. Biol. Chem. 257: pp 2887-2893 (1982)
40.Lambeth, J.D., Tuckey, R. and Kamin, H. "Adrenal Mitochondrial Cytochrome P-450scc: On the Role of Membrane Phospholipids in Cholesterol Side Chain Cleavage." in Microsomes and Drug Oxidations, Fifth International Symposium, (Sato, R. and Kato, R. eds) pp 249-256 (1981). Wiley-Interscience, New York.
41.Tuckey, R.C. and Stevenson, P.M. Triacylglycerols and Glycerophospholipids in Ovaries from Maturing and Superovulated Immature Rats. Aust. J. Biol. Sci. 33: pp 431-440 (1980)
42.Tuckey, R.C. and Stevenson, P.M. Cholesteryl Esterase and Endogenous Cholesteryl Ester Pools in Ovaries from Maturing and Superovulated Immature Rats. Biochim. Biophys. Acta 618: pp 501-509 (1980)
43.Tuckey, R.C. and Stevenson, P.M. Free and Esterified Cholesterol Concentration and Cholesteryl Ester Composition in the Ovaries of Maturing and Superovulated Immature Rats. Biochim. Biophys. Acta 575: pp 46-56 (1979)
44.Tuckey, R.C. and Stevenson, P.M. Methanolysis of Cholesteryl Esters: Conditions for Quantitative Preparation of Methyl Esters. Anal. Biochem. 94: pp 402-408 (1979)
Roles, responsibilities and expertise
Protein Chemistry: Organelle isolation, protein purification, protein association with artificial membranes, chemical labelling, protein digestion and peptide analysis, structure-function analysis.

Enzymology: Cytochrome P450scc catalysis and mechanism, substrate specificity, steady state and rapid reaction kinetics.

Recombinant DNA Technology: Cloning, bacterial expression, site-directed mutagenesis, PCR.

Steroid Biochemistry/Endocrinology: Steroid and lipid extraction and analysis, radioimmunoassay, TLC and GLC analysis of steroids and lipids.

Cell Biology: Placental cell culture
Teaching
Normal Systems 100 (Medicine): Haem, Oxygen and Carbon Dioxide Transport
Biochemistry of the Cell 201 (Science): The Molecules of Life-Chemical Foundations, Lipids and Membranes, Subcellular Organelles, Culture of Eukaryotic Cells
Biochemistry 250 (Agriculture): Fat Metabolism
Molecular Biology 225 (Science): Proteins, Recombinant DNA techniques
Normal Systems 201 (Medicine): Plasma Membrane and Nuclear Receptors, Adrenal Cortex and Steroid Synthesis, Adrenal Medulla, Stress Hormones, Endocrinology of Pregnancy and Parturition, Adrenal Hyperplasias
Molecular and Structural Biochemistry 351 (Science): Protein Chemistry, Cytochrome P450
Graduate Entry Medicine Program: Ovulation, Pregnancy and Parturition
Current projects
Cytochrome P450scc Structure-Function Studies

The conversion of cholesterol to pregnenolone, termed cholesterol side chain cleavage, is the rate-limiting step in steroid hormone synthesis and occurs in the inner mitochondrial membrane. The reaction involves three hydroxylations, all of which occur at a single active site on cytochrome P450scc. Electrons for the hydroxylation reactions are provided by NADPH via a short electron transport chain comprising adrenodoxin reductase and adrenodoxin. The mechanism of the side-chain cleavage reaction has been studied using mitochondria from the human placenta and also purified enzyme. Features of the active site have been identified from catalytic studies using structural analogues of the substrate cholesterol. We have expressed the mature forms of human adrenodoxin reductase and adrenodoxin and bovine and human cytochromes P450scc in E. coli which has provided a convenient source of these enzymes for structure–function studies. Using molecular modeling, cysteine mutagenesis and fluorescent labeling we have determined that the F-G loop of cytochrome P450scc anchors the cytochrome to the phospholipid membrane.
Regulation of Progesterone Synthesis in the Human Placenta

Regulation of the cholesterol-side chain cleavage reaction, which determines the rate of progesterone synthesis by the human placenta, is being investigated. Cholesterol has been found to be saturating for side chain cleavage in freshly isolated trophoblasts but becomes limiting during culture. Studies using sonicated and intact placental mitochondria supplemented with purified adrenodoxin and adrenodoxin reductase have revealed that the activity of cytochrome P450scc in placental mitochondria is limited by electron transport to the cytochrome. Adrenodoxin reductase was confirmed to be the limiting component for electron transport by measuring the steady state concentration of its product, reduced adrenodoxin, during active steroidogenesis by placental mitochondria. Thus the slowest and therefore rate-limiting step in progesterone synthesis is the reduction of adrenodoxin by adrenodoxin reductase. Limiting adrenodoxin reductase results in a substantial pool of oxidized adrenodoxin being present in placental mitochondria during P450scc catalysis. This oxidized adrenodoxin acts as a competitive inhibitor of P450scc and contributes to the low catalytic rate constant observed for cytochrome P450scc in intact placental mitochondria and cultured trophoblasts.
Cholesterol Transport for Steroid Synthesis by the StAR Protein

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in the adrenal cortex, corpus luteum and testis. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P450scc (CYP11A1) converts this cholesterol into pregnenolone. In collaboration with Professor Walt Miller at UCSF we tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P450scc, to acceptor vesicles containing P450scc but no cholesterol, using P450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P450scc activity in acceptor vesicles 5-10 fold following the addition of donor vesicles. Varying the cholesterol content of the donor vesicle had a relatively small effect on the amount of cholesterol transferred by N-62 StAR. These studies show that StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.
Metabolism of Vitamins D2 and D3 and their Precursors by Cytochrome P450scc

In collaboration with Professor Andrzej Slominski at the University of Tennessee, Memphis we tested the ability of P450scc to metabolize vitamins D2 and D3 plus their precursors, ergosterol and 7-dehydrocholesterol. These potential substrates were incubated with purified P450scc and in some cases were also incubated with P450scc in rat adrenal mitochondria. Products were purified by TLC or HPLC and identified by mass spectrometry and/or NMR. We found that human and bovine P450scc cleaves the side chain of the vitamin D3 precursor, 7-dehydrocholesterol, to produce 7-dehydropregnenolone at rates comparable to that seen for cholesterol metabolism. P450scc did not cleave the side chain of vitamin D3 but hydroxylated the side chain producing 20-hydroxyvitamin D3 and 20,22-dihydroxyhydroxyvitamin D3. P450scc converted vitamin D2 to 20-hydroxyvitamin D2 and 17,20-dihydroxvitamin D2, again with no cleavage of the side chain occurring. The major product of ergosterol metabolism was, 17,24-dihydroxyergosterol, with a small amount of mono-hydroxyergosterol also produced. No metabolites were detected when 25-hydroxyvitamin D3 was incubated with cytochrome P450scc. We conclude that P450scc can metabolize vitamin D and its precursors producing novel hydroxylated metabolites with side chain cleavage occurring only for 7-dehydrocholesterol. The cleavage of the side chain of 7-dehydrocholesterol explains the accumulation of 7-dehydrosteroids in Smith-Lemli-Opitz syndrome where there is an excess of 7-dehydrocholesterol due to a 7-dehydrocholesterol reductase deficiency.

Biological testing of these new metabolites of vitamin D is currently underway and preliminary results show some of these compounds are effective inhibitors of skin cell proliferation.
Research profile
Research profile and publications
 

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