V. M. Shkumatov

From structure and functions of steroidogenic enzymes to new technologies of gene engineering

This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.

Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017α

The cDNA encoding cytochrome P-45017α from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactoseinduced yeast cells showed expression of about 240 nmol of P-45017α per liter of the culture. Binding of progesterone to the cytochrome P-45017α was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17α-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017α and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17α-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017α-dependent hydroxylation reaction. The reduction of the 20-keto group of 17α-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017α expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11β- > 11α- > 19-hydroxyprogesterone. In conclusion, the present results show that the host–vector system used is suitable for high-level functional expression of P-45017α and further application of enzymatic properties of this protein to perform specific steroid biotransformations.

Formation and Functioning of Fused Cholesterol Side-Chain Cleavage Enzymes

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.

22-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting oxidoreductases.

Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD-cholesterol), a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH(2)) formation as a dominant fluorescent product. Basing on LC-MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregnenolone and 20-formyl-pregn-5-en-3β-ol were deduced to be steroidal co-products of NBD-NH(2), indicating intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent probe for steroid hormone-binding enzymes or receptors.

Cytochrome P450 Expression in Yarrowia lipolytica and Its Use in Steroid Biotransformation

This review is a first attempt to systematize data on the potential of transgenic Y. lipolytica to catalyze diverse reactions of steroid transformation. The yeast Y. lipolytica was tested as host for P450-catalyzed biotransformation of steroids, including the mammalian P450scc system (three components) and/or the two-component P450c17 system, being functionally active with yeast NADPH-P450 reductase (CPR). New strategies for the construction of recombinant Y. lipolytica strains containing several expression cassettes containing heterologous cDNA (up to 4, in five vectors) under control of the isocitrate lyase (ICL1) promoter have been developed. Characteristics of recombinant Y. lipolytica strains functionally expressing the P450scc system and/or P450c17, being functionally active with yeast NADPH-P450 reductase (YlCPR), are presented.

Evaluation of the fluorescent probes Nile Red and 25‐NBD‐cholesterol as substrates for steroid‐converting oxidoreductases using pure enzymes and microorganisms

The fluorescent probes Nile Red (nonsteroidal dye) and 25‐{N‐[(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐methyl]amino}‐27‐norcholesterol (25‐NBD‐cholesterol) (a cholesterol analog) were evaluated as novel substrates for steroid‐converting oxidoreductases. Docking simulations with autodock showed that Nile Red fits well into the substrate‐binding site of cytochrome P450 17α‐hydroxylase/17,20‐lyase (CYP17A1) (binding energy value of −8.3 kcal·mol−1). Recombinant Saccharomyces cerevisiae and Yarrowia lipolytica, both expressing CYP17A1, were found to catalyze the conversion of Nile Red into two N‐dealkylated derivatives. The conversion by the yeasts was shown to increase in the cases of coexpression of electron‐donating partners of CYP17A1. The highest specific activity value (1.30 ± 0.02 min−1) was achieved for the strain Y. lipolytica DC5, expressing CYP17A1 and the yeasts NADPH‐cytochrome P450 reductase. The dye was also metabolized by pure CYP17A1 into the N‐dealkylated derivatives, and gave a type I difference spectrum when titrated into low‐spin CYP17A1. Analogously, docking simulations demonstrated that 25‐NBD‐cholesterol binds into the active site of the microbial cholesterol oxidase (CHOX) from Brevibacterium sterolicum (binding energy value of −5.6 kcal·mol−1). The steroid was found to be converted into its 4‐en‐3‐one derivative by CHOX (Km and kcat values were estimated to be 58.1 ± 5.9 μm and 0.66 ± 0.14 s−1, respectively). The 4‐en‐3‐one derivative was also detected as the product of 25‐NBD‐cholesterol oxidation with both pure microbial cholesterol dehydrogenase (CHDH) and a pathogenic bacterium, Pseudomonas aeruginosa, possessing CHOXs and CHDHs. These results provide novel opportunities for investigation of the structure–function relationships of the aforementioned oxidoreductases, which catalyze essential steps of steroid bioconversion in mammals (CYP17A1) and bacteria (CHOX and CHDH), with fluorescence‐based techniques.

Range of substrates and steroid bioconversion reactions performed by recombinant microorganisms Saccharomyces cerevisiae and Yarrowia lipolytica expressing cytochrome P450c17

The relationship between 17α-hydroxylation and 20-oxidation-reduction of progesterone and some of its derivatives was studied in yeast strains Saccharomyces cerevisiae YEp51α, Yarrowia lipolytica E129A15, and expressing cytochrome P450c17. The key metabolites were found to be 17α-hydroxyprogester-one and 17α,20(α,β)-dihydroxypregn-4-ene-3-ones. The bioconversion pathways of pregn-4-ene-20(α,β)-ol-3-ones were determined. They included cycles of 20-oxidation, 17α-hydroxylation, and stereospecific 20-reduction. The efficiency and kinetic parameters of steroid bioconversion by the recombinant strains were determined. The role of yeast analogs of mammalian steroid dehydrogenases is discussed. It was found that any of the desired derivatives, 17α-hydroxyprogesterone or progesterone 17α,20(α,β)-diols, could be obtained from progesterone. Cholesterol bioconversion yields important metabolites: steroid hormones, the vitamin-D group, and bile acids [1, 2]. Attention to various cytochrome-P450 species participating in the biosynthesis of mammalian steroid hormones is caused by two circumstances: (1) the necessity of detecting structural-function abnorm alities of some of the enzymes of steroid-synthesis that cause human diseases, and (2) the potential of regio-and stereospecific cytochrome P450 species of mammals in chemoenzymatic synthesis of pharmacologically valuable steroids. Concerning the second line of inquiry, the development of transgenic Saccharomyces cerevisiae yeast for the complete synthesis of cortisol by additional expression and elimination of a total of 13 genes was reported [3]. To increase the yield of the target compound, the genes for enzymes performing undesirable steroid modifications were inactivated. These modifications included esterification of pregnenolone [4] and 20α-reduction of 17α-hydroxyprogesterone [5]. A search for analogs of mammalian 20α-hydroxysteroid dehydrogenase (20α-HSD) in the Saccharomyces cerevisiae genome revealed two candidate proteins: Ypr1p (yeast aldo-keto reductase) and Gcy1p (yeast galactose-inducible crystallin-like protein) [3]. Indeed, it was formerly shown that expression of cytochrome P450 from bovine adrenal cortex, performing 17α-hydroxylation and the C17,20-lyase reaction (P450c17) in S. cerevisiae under the control of the GAL10-promoter with the presence of D-galactose as an inducer, was accompanied by the sequential conversion of progesterone to 17α-hydroxyprogesterone and 17α,20(α,β)-dihydroxypregn-4-ene-3-one with a high yield [5].

22-NBD-cholesterol, a new fluorescent substrate of bacterial cholesterol-oxidases

Fluorescent analogs of sterols are convenient agents for studying the transport, distribution, and protein—ligand interaction of natural sterols in cells [1]. However, the effect of the fluorescent moiety on the interaction parameters with cholesterol-transforming enzymes such as bacterial cholesterol-oxidases or mammalian steroid-hydroxylating cytochrome P450 has not been addressed in the literature. Herein we report data for the interaction of the fluorescent cholesterol analog 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3 -ol [22-NBD-cholesterol (1)] with cholesteroloxidases (EC 1.1.3.6). Cholesterol-oxidases are FAD-dependent enzymes that catalyze the transformation of 3 -hydroxy5steroids and are widely used for laboratory and clinical cholesterol determinations [2]. We calculated the interaction of 1 with the enzyme based on the three-dimensional structure of Brevibacterium sterolicum cholesterol-oxidase (code PDB 1COY) using the Autodock 4.0 programs and MGLTools. It was found with the help of molecular docking that 1 can bind to the active center of cholesterol-oxidase such that its 3 -hydroxy group is situated in close proximity to the functionally important His447 and Glu361 residues [3] and the redox-active groups of the FAD molecule. The calculated binding energies for cholesterol and 1 were practically the same (–11.43 and –11.94 kcal/mol, respectively). Next selective transformation of 1 was demonstrated using cholesterol-oxidase in vitro. After biotransformation of fluorescent substrate 1 with purified cholesterol-oxidase, the formation of 2, which retained the fluorescent properties of 1 (Scheme 1) was detected by HPLC (Shimadzu LC10-AD chromatograph with an RF-10A 1 fluorimetric detector and SPD-10A spectrophotometric detector; CH3CN:H2O:i-PrOH, 84:16:5; LiChroCART C18 column, Merck, 250 4 mm).

Effects of low frequency ultrasound on some properties of fibrinogen and its plasminolysis

BackgroundPharmacological thrombolysis with streptokinase, urokinase or tissue activator of plasminogen (t-PA), and mechanical interventions are frequently used in the treatment of both arterial and venous thrombotic diseases. It has been previously reported that application of ultrasound as an adjunct to thrombolytic therapy offers unique potential to improve effectiveness. However, little is known about effects of the ultrasound on proteins of blood coagulation and fibrinolysis. Here, we investigated the effects of the ultrasound on fibrinogen on processes of coagulation and fibrinogenolysis in an in vitro system.ResultsOur study demonstrated that low frequency high intensity pulse ultrasound (25.1 kHz, 48.4 W/cm2, duty 50%) induced denaturation of plasminogen and t-PA and fibrinogen aggregates formation in vitro. The aggregates were characterized by the loss of clotting ability and a greater rate of plasminolysis than native fibrinogen. We investigated the effect of the ultrasound on individual proteins. In case of plasminogen and t-PA, ultrasound led to a decrease of the fibrinogenolysis rate, while it increased the fibrinogenolysis rate in case of fibrinogen. It has been shown that upon ultrasound treatment of mixture fibrinogen or fibrin with plasminogen, t-PA, or both, the rate of proteolytic digestion of fibrin(ogen) increases too. It has been shown that summary effect on the fibrin(ogen) proteolytic degradation under the conditions for combined ultrasound treatment is determined exclusively by effect on fibrin(ogen).ConclusionsThe data presented here suggest that among proteins of fibrinolytic systems, the fibrinogen is one of the most sensitive proteins to the action of ultrasound. It has been shown in vitro that ultrasound induced fibrinogen aggregates formation, characterized by the loss of clotting ability and a greater rate of plasminolysis than native fibrinogen in different model systems and under different mode of ultrasound treatment. Under ultrasound treatment of plasminogen and/or t-PA in the presence of fibrin(ogen) the stabilizing effect fibrin(ogen) on given proteins was shown. On the other hand, an increase in the rate of fibrin(ogen) lysis was observed due to both the change in the substrate structure and promoting of the protein-protein complexes formation.

Expression of cytochrome P450scc in Escherichia coli cells: Intracellular location and interaction with bacterial redox proteins

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.