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Rita Bernhardt - One of the best experts on this subject based on the ideXlab platform.

  • cyp109e1 from bacillus megaterium acts as a 24 and 25 hydroxylase for cholesterol
    ChemBioChem, 2019
    Co-Authors: Natalia Putkaradze, Martin Litzenburger, Michael C Hutter, Rita Bernhardt
    Abstract:

    : In this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with Adrenodoxin and Adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole-cell system that harbored CYP109E1 was established. This novel system showed a 3.3-fold higher activity than that of the B. megaterium system, yielding about 45 mg L-1 of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)- and 25-hydroxycholesterol.

  • Human CYP27A1 catalyzes hydroxylation of β-sitosterol and ergosterol.
    Biological Chemistry, 2016
    Co-Authors: Maximilian Ehrhardt, Adrian Gerber, Frank Hannemann, Josef Zapp, Rita Bernhardt
    Abstract:

    : β-Sitosterol and ergosterol are the equivalents of cholesterol in plants and fungi, respectively, and common sterols in the human diet. In the current work, both were identified as novel CYP27A1 substrates by in vitro experiments applying purified human CYP27A1 and its redox partners Adrenodoxin (Adx) and Adrenodoxin reductase (AdR). A Bacillus megaterium based biocatalyst recombinantly expressing the same proteins was utilized for the conversion of the substrates to obtain sufficient amounts of the novel products for a structural NMR analysis. β-Sitosterol was found to be converted into 26-hydroxy-β-sitosterol and 29-hydroxy-β-sitosterol, whereas ergosterol was converted into 24-hydroxyergosterol, 26-hydroxyergosterol and 28-hydroxyergosterol.

  • Dehydroepiandrosterone Sulfate (DHEAS) Stimulates the First Step in the Biosynthesis of Steroid Hormones
    2014
    Co-Authors: Jens Neunzig, Rita Bernhardt
    Abstract:

    Dehydroepiandrosterone sulfate (DHEAS) is the most abundant circulating steroid in human, with the highest concentrations between age 20 and 30, but displaying a significant decrease with age. Many beneficial functions are ascribed to DHEAS. Nevertheless, long-term studies are very scarce concerning the intake of DHEAS over several years, and molecular investigations on DHEAS action are missing so far. In this study, the role of DHEAS on the first and rate-limiting step of steroid hormone biosynthesis was analyzed in a reconstituted in vitro system, consisting of purified CYP11A1, Adrenodoxin and Adrenodoxin reductase. DHEAS enhances the conversion of cholesterol by 26%. Detailed analyses of the mechanism of DHEAS action revealed increased binding affinity of cholesterol to CYP11A1 and enforced interaction with the electron transfer partner, Adrenodoxin. Difference spectroscopy showed Kd-values of 4062.7 mM and 24.860.5 mM for CYP11A1 and cholesterol without and with addition of DHEAS, respectively. To determine the Kd-value for CYP11A1 and Adrenodoxin, surface plasmon resonance measurements were performed, demonstrating a Kd-value of 3.060.35 nM (with cholesterol) and of 2.460.05 nM when cholesterol and DHEAS were added. Kinetic experiments showed a lower Km and a higher kcat value for CYP11A1 in the presence of DHEAS leading to an increase of the catalytic efficiency by 75%. These findings indicate that DHEAS affects steroid hormone biosynthesis on a molecular level resulting in an increased formation of pregnenolone

  • Adrenodoxin the archetype of vertebrate type 2fe 2s cluster ferredoxins proteins and proteomics
    Biochimica et Biophysica Acta, 2011
    Co-Authors: Kerstin Maria Ewen, Michael Kleser, Rita Bernhardt
    Abstract:

    Adrenodoxin is probably the best characterized member of the vertebrate-type [2Fe–2S]-cluster ferredoxins. It has been in the spotlight of scientific interest for many years due to its essential role in mammalian steroid hormone biosynthesis, where it acts as electron mediator between the NADPH-dependent Adrenodoxin reductase and several mitochondrial cytochromes P450. In this review we will focus on the present knowledge about protein–protein recognition in the mitochondrial cytochrome P450 system and the modulation of the electron transfer between Adx and its redox partners, AdR and CYP(s). We also intend to point out the potential biotechnological applications of Adx as a versatile electron donor to different cytochromes P450, both in vitro and in vivo. Finally we will address the comparison between the mammalian cytochrome P450-associated Adrenodoxin and ferredoxins involved in iron–sulfur-cluster biosynthesis. Despite their different functions, these proteins display an amazing similarity regarding their primary sequence, tertiary structure and biophysical features.

  • Adrenodoxin the archetype of vertebrate type 2fe 2s cluster ferredoxins
    Biochimica et Biophysica Acta, 2011
    Co-Authors: Kerstin Maria Ewen, Michael Kleser, Rita Bernhardt
    Abstract:

    Abstract Adrenodoxin is probably the best characterized member of the vertebrate-type [2Fe–2S]-cluster ferredoxins. It has been in the spotlight of scientific interest for many years due to its essential role in mammalian steroid hormone biosynthesis, where it acts as electron mediator between the NADPH-dependent Adrenodoxin reductase and several mitochondrial cytochromes P450. In this review we will focus on the present knowledge about protein–protein recognition in the mitochondrial cytochrome P450 system and the modulation of the electron transfer between Adx and its redox partners, AdR and CYP(s). We also intend to point out the potential biotechnological applications of Adx as a versatile electron donor to different cytochromes P450, both in vitro and in vivo. Finally we will address the comparison between the mammalian cytochrome P450-associated Adrenodoxin and ferredoxins involved in iron–sulfur-cluster biosynthesis. Despite their different functions, these proteins display an amazing similarity regarding their primary sequence, tertiary structure and biophysical features.

Michael R. Waterman - One of the best experts on this subject based on the ideXlab platform.

  • an additional electrostatic interaction between Adrenodoxin and p450c27 cyp27a1 results in tighter binding than between Adrenodoxin and p450scc cyp11a1
    Journal of Biological Chemistry, 1999
    Co-Authors: Irina A Pikuleva, Carolyn Cao, Michael R. Waterman
    Abstract:

    Abstract Mitochondrial cytochrome P450c27 (product of the CYP27A1 gene) is found to have significantly higher affinity for the common redox partner Adrenodoxin than another mitochondrial P450, P450scc (product of the CYP11A1 gene). To investigate the basis of the ∼30-fold difference in Adrenodoxin binding, two sets of P450c27 mutants were generated, expressed in Escherichia coli, and purified. Mutations of one set were within the putative Adrenodoxin-binding site containing conserved lysine residues also crucial in P450scc for binding Adrenodoxin. The second set included mutations within a sequence aligning with the “meander region” of P450BM-3 proposed to be a site of redox-partner interactions in P450s (Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure3, 41–62). Mutation of the P450c27 conserved lysines (K354A and K358A) led to a ∼20-fold increase in apparent K s for Adrenodoxin, confirming that these two positively charged residues conserved in mitochondrial P450s are important for Adrenodoxin binding. Mutation of Arg-418, conserved in the CYP27A1 family, to serine also decreased the affinity for Adrenodoxin ∼20-fold. This residue is predicted to be located in the meander region. A triple K354A/K358A/R418S mutation profoundly reduced Adrenodoxin binding. Thus, in contrast to P450scc, where mutation of the two conserved positively charged residues results in virtually complete inhibition of Adrenodoxin binding, in P450c27 there are three of such residues (Lys-354, Lys-358, and Arg-418) important for Adrenodoxin interaction.

  • Direct expression of Adrenodoxin reductase in Escherichia coli and the functional characterization.
    Biological & pharmaceutical bulletin, 1993
    Co-Authors: Yasuhiro Sagara, Michael R. Waterman, Yutaka Takata, Kazuhisa Sekimizu, Akira Wada, Tadao Horiuchi
    Abstract:

    A plasmid for direct expression in Escherichia coli of the mature form bovine Adrenodoxin reductase was constructed from the full-size cDNA for the enzyme [Y. Sagara, Y. Takata, T. Miyata, T. Hara, and T. Horiuchi, J. Biochem. (Tokyo), 102, 1333 (1987)] and an expression vector pCWori+. The recombinant Adrenodoxin reductase was purified from the transformed E. coli cell lysates using Adrenodoxin-Sepharose affinity chromatography [T. Sugiyama and T. Yamano, FEBS Lett., 52, 145 (1975)] with a yield of 2.5 mg/l of culture. The purified recombinant enzyme showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and migration was identical with that of the authentic enzyme purified from bovine adrenal cortex mitochondria. The recombinant enzyme had Ser at its amino-terminus and the sequence of the amino terminal 9 residues was identical with that of the authentic bovine enzyme. The absorption spectrum of the recombinant enzyme showed peaks at 270, 376, and 450 nm and shoulders at 425 and 475 nm. Flavin content of the recombinant enzyme was 0.8 mol FAD/mol. The apparent Km value for bovine Adrenodoxin in NADPH-cytochrome c reductase activity using a reconstitution system was 16 nM, a value comparable with that of the authentic bovine enzyme (17 nM). The cholesterol side chain cleavage activity with a reconstitution system was about 75% of that obtained when the authentic enzyme was used.

  • identification by site directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome p450 that are essential for Adrenodoxin binding
    Journal of Biological Chemistry, 1992
    Co-Authors: A Wada, Michael R. Waterman
    Abstract:

    Utilizing site-directed mutagenesis and an Escherichia coli expression system for bovine cholesterol side chain cleavage cytochrome P450, lysine residues at 377 and 381 are found to play crucial roles in binding bovine Adrenodoxin, required for transfer of electrons to mitochondrial P450s. These lysine residues are conserved among mitochondrial P450s and have been implicated previously by chemical modification studies as being important for Adrenodoxin binding. In the present study, site-directed mutagenesis producing either neutral or positive amino acids at 377 or 381 has no effect on the structure of side chain cleavage cytochrome P450 as determined spectrally or on the enzymatic conversion of cholesterol to pregnenolone. However, the estimated Ks of Adrenodoxin binding is increased approximately 150-600-fold depending on the particular mutation. Therefore these conserved positively charged residues in mitochondrial P450s are the key sites for Adrenodoxin binding which is electrostatic in nature.

  • direct expression of mature bovine Adrenodoxin in escherichia coli
    Archives of Biochemistry and Biophysics, 1992
    Co-Authors: Mariefrance Palin, Michael R. Waterman, Luc Berthiaume, Jeanguy Lehoux, Jurgen Sygusch
    Abstract:

    Abstract Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine Adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli . Expression was under control of the “tac” promoter and resulted in a direct expression of soluble mature bovine Adrenodoxin (>15 mg per liter). Chromatographic behavior of recombinant Adrenodoxin did not differ from that reported for mature native Adrenodoxin. The purified recombinant protein was identical to native mitochondrial Adrenodoxin on the basis of molecular weight, NH 2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine Adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo . Recombinant bovine Adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with Adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine Adrenodoxin. The presence of the Adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant Adrenodoxin in E. coli . This expression system should prove useful for overexpression of Adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.

  • two promoters in the bovine Adrenodoxin gene and the role of associated unique camp responsive sequences
    Biochemistry, 1992
    Co-Authors: Jeou Yuan Chen, Michael R. Waterman
    Abstract:

    The bovine Adrenodoxin gene gives rise to two species of mRNA differing only at their 5'-ends. The synthesis of these two types of mRNA in bovine adrenal cortical cells is regulated transcriptionally in part by ACTH via the action of cAMP. Examination of the 5'-end of the Adrenodoxin gene revealed that each mRNA contains sequences derived from a different exon encoding the mitochondrial leader sequence. To define the sequences necessary for the synthesis of these two types of mRNA and to determine if the synthesis of each is driven by a separate promoter, 5'-regions of the Adrenodoxin gene were inserted upstream from two different reporter genes and tested for promoter/enhancer regulatory activity by transient transfection into mouse adrenocortical Y1 tumor cells. The results clearly demonstrated that the bovine Adrenodoxin gene contains two functional promoters; one, ADXP1, located in the 5'-flanking region gives rise to the minor form of mRNA, and a second, stronger promoter, ADXP2, which maps within intron 1 gives rise to the major form of mRNA. Unique cAMP-responsive sequences were found upstream from each promoter which share no sequence homology to the consensus CRE (cAMP-responsive element). Upon transient expression, the cAMP-responsive sequence associated with the ADXP2 promoter, termed CRS2, confers the cAMP responsiveness to stimulate the transcription of the linked beta-globin reporter gene regardless of whether the Adrenodoxin ADXP2 promoter or the beta-globin promoter was utilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Frank Hannemann - One of the best experts on this subject based on the ideXlab platform.

  • Human CYP27A1 catalyzes hydroxylation of β-sitosterol and ergosterol.
    Biological Chemistry, 2016
    Co-Authors: Maximilian Ehrhardt, Adrian Gerber, Frank Hannemann, Josef Zapp, Rita Bernhardt
    Abstract:

    : β-Sitosterol and ergosterol are the equivalents of cholesterol in plants and fungi, respectively, and common sterols in the human diet. In the current work, both were identified as novel CYP27A1 substrates by in vitro experiments applying purified human CYP27A1 and its redox partners Adrenodoxin (Adx) and Adrenodoxin reductase (AdR). A Bacillus megaterium based biocatalyst recombinantly expressing the same proteins was utilized for the conversion of the substrates to obtain sufficient amounts of the novel products for a structural NMR analysis. β-Sitosterol was found to be converted into 26-hydroxy-β-sitosterol and 29-hydroxy-β-sitosterol, whereas ergosterol was converted into 24-hydroxyergosterol, 26-hydroxyergosterol and 28-hydroxyergosterol.

  • Structural and Thermodynamic Characterization of the Adrenodoxin-Like Domain of the Electron-Transfer Protein Etp1 from Schizosaccharomyces Pombe.
    Journal of Inorganic Biochemistry, 2011
    Co-Authors: Jürgen Müller, Frank Hannemann, Burkhard Schiffler, Kerstin Maria Ewen, Reinhard Kappl, Udo Heinemann, Rita Bernhardt
    Abstract:

    The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1(fd), which is cleaved off after mitochondrial import. The physiological function of Etp1(fd) is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, Adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1(fd) has a melting temperature more than 20°C higher than Adrenodoxin. The three-dimensional structure of Etp1(fd) has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1(fd) with Adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1(fd) and Adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.

  • the dipole moment of the electron carrier Adrenodoxin is not critical for redox partner interaction and electron transfer
    Journal of Inorganic Biochemistry, 2009
    Co-Authors: Frank Hannemann, Udo Heinemann, Arnaud Guyot, Andy Zollner, Jurgen J Muller, Rita Bernhardt
    Abstract:

    Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein Adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine Adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length Adrenodoxin model based on the Adx(4-108) and the wild type Adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant Adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified Adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.

  • The endogenous Adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.
    FEMS yeast research, 2008
    Co-Authors: Kerstin Maria Ewen, Burkhard Schiffler, Rita Bernhardt, Heike Uhlmann-schiffler, Frank Hannemann
    Abstract:

    Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via Adrenodoxin and Adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog Adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

  • deletions in the loop surrounding the iron sulfur cluster of Adrenodoxin severely affect the interactions with its native redox partners Adrenodoxin reductase and cytochrome p450scc cyp11a1
    Journal of Inorganic Biochemistry, 2002
    Co-Authors: Andy Zollner, Frank Hannemann, Michael Lisurek, Rita Bernhardt
    Abstract:

    The redox active iron-sulfur center of bovine Adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe-2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe-2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in Adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners Adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein.

Udo Heinemann - One of the best experts on this subject based on the ideXlab platform.

  • Structural and Thermodynamic Characterization of the Adrenodoxin-Like Domain of the Electron-Transfer Protein Etp1 from Schizosaccharomyces Pombe.
    Journal of Inorganic Biochemistry, 2011
    Co-Authors: Jürgen Müller, Frank Hannemann, Burkhard Schiffler, Kerstin Maria Ewen, Reinhard Kappl, Udo Heinemann, Rita Bernhardt
    Abstract:

    The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1(fd), which is cleaved off after mitochondrial import. The physiological function of Etp1(fd) is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, Adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1(fd) has a melting temperature more than 20°C higher than Adrenodoxin. The three-dimensional structure of Etp1(fd) has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1(fd) with Adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1(fd) and Adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.

  • the dipole moment of the electron carrier Adrenodoxin is not critical for redox partner interaction and electron transfer
    Journal of Inorganic Biochemistry, 2009
    Co-Authors: Frank Hannemann, Udo Heinemann, Arnaud Guyot, Andy Zollner, Jurgen J Muller, Rita Bernhardt
    Abstract:

    Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein Adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine Adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length Adrenodoxin model based on the Adx(4-108) and the wild type Adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant Adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified Adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.

  • covalently crosslinked complexes of bovine Adrenodoxin with Adrenodoxin reductase and cytochrome p450scc
    FEBS Journal, 2001
    Co-Authors: Evachristina Muller, Jürgen Müller, Anna Lapko, Albrecht Otto, Klaus Ruckpaul, Udo Heinemann
    Abstract:

    NADPH-dependent Adrenodoxin reductase, Adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, Adrenodoxin transfers electrons from the FAD of Adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for Adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from Adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine Adrenodoxin and Adrenodoxin reductase, and between Adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between Adrenodoxin reductase Lys27 and Adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of Adrenodoxin. However, in a covalent complex of Adrenodoxin and P450scc, Adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an Adrenodoxin −P450scc complex/Adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of Adrenodoxin to Adrenodoxin reductase, as well as to the P450scc, the covalent bond within the Adrenodoxin−P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.

  • Covalently crosslinked complexes of bovine Adrenodoxin with Adrenodoxin reductase and cytochrome P450scc. Mass spectrometry and Edman degradation of complexes of the steroidogenic hydroxylase system.
    European journal of biochemistry, 2001
    Co-Authors: Evachristina Muller, Jürgen Müller, Anna Lapko, Albrecht Otto, Klaus Ruckpaul, Udo Heinemann
    Abstract:

    NADPH-dependent Adrenodoxin reductase, Adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, Adrenodoxin transfers electrons from the FAD of Adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for Adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from Adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine Adrenodoxin and Adrenodoxin reductase, and between Adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between Adrenodoxin reductase Lys27 and Adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of Adrenodoxin. However, in a covalent complex of Adrenodoxin and P450scc, Adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an Adrenodoxin -P450scc complex/Adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of Adrenodoxin to Adrenodoxin reductase, as well as to the P450scc, the covalent bond within the Adrenodoxin-P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.

  • Adrenodoxin structure stability and electron transfer properties
    Proteins, 2000
    Co-Authors: Asya Grinberg, Frank Hannemann, Jürgen Müller, Burkhard Schiffler, Udo Heinemann, Rita Bernhardt
    Abstract:

    Adrenodoxin is an iron-sulfur protein that belongs to the broad family of the [2Fe-2S]-type ferredoxins found in plants, animals and bacteria. Its primary function as a soluble electron carrier between the NADPH-dependent Adrenodoxin reductase and several cytochromes P450 makes it an irreplaceable component of the steroid hormones biosynthesis in the adrenal mitochondria of vertebrates. This review intends to summarize current knowledge about structure, function, and biochemical behavior of this electron transferring protein. We discuss the recently solved first crystal structure of the vertebrate-type ferredoxin, the truncated Adrenodoxin Adx(4-108), that offers the unique opportunity for better understanding of the structure-function relationships and stabilization of this protein, as well as of the molecular architecture of [2Fe-2S] ferredoxins in general. The aim of this review is also to discuss molecular requirements for the formation of the electron transfer complex. Essential comparison between bacterial putidaredoxin and mammalian Adrenodoxin will be provided. These proteins have similar tertiary structure, but show remarkable specificity for interactions only with their own cognate cytochrome P450. The discussion will be largely centered on the protein-protein recognition and kinetics of Adrenodoxin dependent reactions. Proteins 2000;40:590–612. © 2000 Wiley-Liss, Inc.

Robert C. Tuckey - One of the best experts on this subject based on the ideXlab platform.

  • Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating Adrenodoxin reductase.
    The Journal of steroid biochemistry and molecular biology, 2002
    Co-Authors: Robert C. Tuckey, Madeleine J. Headlam
    Abstract:

    Abstract The mitochondrial side-chain cleavage of cholesterol, catalysed by cytochrome P450scc, is rate-limiting in the synthesis of progesterone by the human placenta. Cytochrome P450scc activity is in turn limited by the concentration of Adrenodoxin reductase (AR) in placental mitochondria. In order to better understand which components of the cholesterol side-chain cleavage system are important in the regulation of placental progesterone synthesis, we have examined their effects on P450scc activity with both saturating and limiting concentrations of AR. The present study reveals that decreasing the AR concentration causes a decrease in the K m of cytochrome P450scc for cholesterol, facilitating saturation of the enzyme with its substrate. Decreasing AR resulted in P450scc activity becoming less sensitive to changes in P450scc concentration. The Adrenodoxin (Adx) concentration in mitochondria from term placentae is near-saturating for P450scc and under these conditions, we found that decreasing AR reduces the K m of P450scc for Adrenodoxin. Increasing either the cholesterol or P450scc concentration increased the amount of AR required for P450scc to work at half its maximum velocity. A relatively small increase in AR can support considerably higher rates of side-chain cleavage activity when there is a coordinate increase in AR and P450scc concentrations. We conclude from this study that cholesterol is near-saturating for cytochrome P450scc activity in placental mitochondria due to the P450scc displaying a low K m for cholesterol resulting from the low and rate-limiting concentration of AR present. This study reveals that it is unlikely that cholesterol or Adrenodoxin concentrations are important regulators of placental progesterone synthesis but AR or coordinate changes in AR and P450scc concentrations are likely to be important in its regulation.

  • Oxidized Adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta
    European journal of biochemistry, 2001
    Co-Authors: Robert C. Tuckey, Allan J. Mckinley, Madeleine J. Headlam
    Abstract:

    The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate-determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of Adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce Adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of Adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the Adrenodoxin pool was only 30% reduced, indicating that the Adrenodoxin reductase concentration was insufficient to maintain the Adrenodoxin in the fully reduced state. As both oxidized and reduced Adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized Adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified Adrenodoxin and Adrenodoxin reductase. We found that oxidized Adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced Adrenodoxin. We conclude that the limiting concentration of Adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced Adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized Adrenodoxin that remains, competitively inhibits the cytochrome.

  • The concentration of Adrenodoxin reductase limits cytochrome P450scc activity in the human placenta
    European journal of biochemistry, 1999
    Co-Authors: Robert C. Tuckey, Jade Sadleir
    Abstract:

    We have previously reported that cytochrome P450scc activity in the human placenta is limited by the supply of electrons to the P450scc [Tuckey, R. C., Woods, S. T. & Tajbakhsh, M. (1997) Eur. J. Biochem. 244, 835‐839]. The aim of the present study was to determine whether it is Adrenodoxin reductase, Adrenodoxin or both which limits cytochrome P450scc activity and hence progesterone synthesis in the placenta. We found that the concentrations of Adrenodoxin reductase and Adrenodoxin in placental mitochondria were both considerably lower than the concentrations of these proteins in the bovine adrenal cortex. When P450scc activity assays were carried out at high mitochondrial protein concentrations, we found that the addition of exogenous Adrenodoxin reductase to sonicated mitochondria rescued pregnenolone synthesis to a level above that for intact mitochondria, showing that Adrenodoxin is near-saturating in vivo. In contrast, pregnenolone synthesis by sonicated mitochondria was almost zero even after the addition of human Adrenodoxin. This shows that the concentration of endogenous Adrenodoxin reductase was insufficient to support appreciable rates of pregnenolone synthesis, even when concentrated mitochondrial samples were used. Comparative studies with human and bovine Adrenodoxin reductase have revealed that a twofold higher concentration of human Adrenodoxin reductase is required for maximal P450scc activity in the presence of saturating human Adrenodoxin. Thus, not only is the Adrenodoxin concentration low in placental mitochondria, but the amount required for maximal P450scc activity is higher than that for the bovine reductase. Overall, the data indicate that the Adrenodoxin reductase concentration limits the activity of P450scc in placental mitochondria and hence determines the rate of progesterone synthesis.

  • 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
    FEBS Journal, 1999
    Co-Authors: Pyotr Kisselev, Robert C. Tuckey, Stephen T Woods, Thrassi Triantopoulos, Dieter Schwarz
    Abstract:

    The recently reported heterologous expression and purification of both human cytochrome P450SCC and Adrenodoxin [Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Biophys. 353, 109-115] has enabled us to perform studies with the membrane-reconstituted human enzymes to better understand the side-chain cleavage reaction in humans. Human P450SCC was successfully reconstituted into dioleoylphosphatidylcholine vesicles with and without cardiolipin and its enzymatic properties characterized in the membrane-bound state. Enhancement of the P450SCC activity and significant activation by cardiolipin were observed when human Adrenodoxin instead of bovine Adrenodoxin was used as electron donor. In the absence of cardiolipin, Km for cholesterol was decreased twice in the case of human Adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both Km and V for cholesterol were decreased with human Adrenodoxin as electron donor. Kinetic analysis of the interaction between human P450SCC and its redox partners provided evidence for enhanced binding of the human electron donor to human P450SCC indicated by both an increased V and decreased Kd for human Adrenodoxin compared with the values with bovine Adrenodoxin. Because no similar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human Adrenodoxin with human P450SCC.