Publications

• Engineering, expression, and purification of "soluble" human cytochrome P45017alpha and its functional characterization.

  Pechurskaya T.A., Lukashevich O.P., Gilep A.A., Usanov S.A.

  To engineer a "soluble" form of membrane-bound cytochrome P45017alpha (CYP17)--a key enzyme in steroid hormone biosynthesis--in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these re ... >> More

  To engineer a "soluble" form of membrane-bound cytochrome P45017alpha (CYP17)--a key enzyme in steroid hormone biosynthesis--in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (Delta(23))) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane. However, CYP17mod containing hydrophilic FG-loop is mostly localized in the cytosolic fraction. Thus, in the present work we for the first time engineered a "soluble" form of the usually membrane-bound human CYP17 that is not bound to membrane. The expression degree of CYP17mod is approximately 900 nmol/liter of culture. The hemeprotein can be purified to apparent homogeneity without using detergents at any purification step. It is shown that replacement of hydrophobic amino acid residues in the FG-loop region does not change the metabolic profile during hydroxylation of steroids that is characteristic for wild type CYP17. Besides, the modification of the hemeprotein does not affect the affinity of CYP17 to steroid substrates. The engineered "soluble" form of human CYP17 is used as a subject for crystallization of the hemeprotein. << Less

  Biochemistry (Mosc) 73:806-811(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular cloning and heterologous expression in E. coli of cytochrome P45017alpha. Comparison of structural and functional properties of substrate-specific cytochromes P450 from different species.

  Gilep A.A., Estabrook R.W., Usanov S.A.

  To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017alpha from species of the Bovidae family (sheep, goat, and bison), ... >> More

  To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017alpha from species of the Bovidae family (sheep, goat, and bison), which catalyze 17alpha-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C(19)-steroids. Recombinant cytochromes P45017alpha were expressed in E. coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography. Highly purified cytochromes P45017alpha were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17alpha-hydroxyprogesterone, and 17alpha-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC. It is shown that each form of cytochrome P45017alpha is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b(5) in the reaction mixture. The analysis of the activity of the known forms of cytochrome P45017alpha in view of the data obtained in the present work allows the division of known cytochromes P45017alpha into three main group: group A (pig, hamster, rat), cytochromes P45017alpha catalyze the reaction of 17alpha-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17alpha-hydroxyprogesterone and 17alpha-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017alpha, which have no or have insignificant 17,20-lyase activity in relation to 17alpha-hydroxyprogesterone; group C (guinea pig), cytochrome P45017alpha which either has no or has insignificant 17,20-lyase activity on transformation 17alpha-hydroxypregnenolone to dehydroepiandrosterone. << Less

  Biochemistry (Mosc.) 68:86-98(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The conversion of progesterone to androgens by testes.

  LYNN W.S. Jr., BROWN R.H.

  J Biol Chem 232:1015-1030(1958) [PubMed] [EuropePMC]

• Functional expression and characterisation of human cytochrome P45017alpha in Pichia pastoris.

  Kolar N.W., Swart A.C., Mason J.I., Swart P.

  Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initiall ... >> More

  Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17. The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17. This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris. << Less

  J Biotechnol 129:635-644(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Studies of the human testis. XIII. Properties of nicotinamide adenine dinucleotide (reduced form)-linked 17 alpha-hydroxylation.

  Yoshida K.I., Oshima H., Troen P.

  A NADH-linked 17 alpha-hydroxylation of progesterone was examined using the washed microsome fraction prepared from human testes. The addition of NADP revealed no enhancement of 17 alpha-hydroxylation, indicating no transhydrogenation from NADH to NADP in the microsome fraction. The results furthe ... >> More

  A NADH-linked 17 alpha-hydroxylation of progesterone was examined using the washed microsome fraction prepared from human testes. The addition of NADP revealed no enhancement of 17 alpha-hydroxylation, indicating no transhydrogenation from NADH to NADP in the microsome fraction. The results further substantiate the presence of NADH-linked activity of 17 alpha-hydroxylation in addition to NADPH-linked activity. The Km of 17 alpha-hydroxylase for NADH was calculated as 4.3 x 10(-5) M at pH 7.4 and 37 C. The optimal pH of 17 alpha-hydroxylase was 7.7 in the presence of NADH and 7.9 in the presence of NADPH. An additive increase in the amount of product 17 alpha-hydroxyprogesterone was observed by adding NADH to the incubation medium containing an excess amount of NADPH. The data suggest that there are two distinct active sites for 17 alpha-hydroxylation. Furthermore, the inhibition of NADH-linked 17 alpha-hydroxylation by carbon monoxide indicates the involvement of cytochrome P450 in the electron transport system for the NADH-linked 17 alpha-hydroxylation. << Less

  J Clin Endocrinol Metab 50:895-899(1980) [PubMed] [EuropePMC]