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Israel Hanukoglu – One of the best experts on this subject based on the ideXlab platform.

  • Conservation of the Enzyme–Coenzyme Interfaces in FAD and NADP Binding Adrenodoxin Reductase—A Ubiquitous Enzyme
    Journal of Molecular Evolution, 2017
    Co-Authors: Israel Hanukoglu
    Abstract:

    FAD and NAD(P) together represent an ideal pair for coupled redox reactions in their capacity to accept two electrons and their redox potentials. Enzymes that bind both NAD(P) and FAD represent large superfamilies that fulfill essential roles in numerous metabolic pathways. Adrenodoxin Reductase (AdxR) shares Rossmann fold features with some of these superfamilies but remains in a group of its own in the absence of sequence homology. This article documents the phylogenetic distribution of AdxR by examining whole genome databases for Metazoa, Plantae, Fungi, and Protista, and determines the conserved structural features of AdxR. Scanning these databases showed that most organisms have a single gene coding for an AdxR ortholog. The sequence identity between AdxR orthologs is correlated with the phylogenetic distance among metazoan species. The NADP binding site of all AdxR orthologs showed a modified Rossmann fold motif with a GxGxxA consensus instead of the classical GxGxxG at the edge of the first βα -fold. To examine the hypothesis that enzyme–coenzyme interfaces represent the conserved regions of AdxR, the residues interfacing FAD and NADP were identified and compared with multiple-sequence alignment results. Most conserved residues were indeed found at sites that surround the interfacing residues between the enzyme and the two coenzymes. In contrast to proteinprotein interaction hot-spots that may appear in isolated patches, in AdxR the conserved regions show strict preservation of the overall structure. This structure maintains the precise positioning of the two coenzymes for optimal electron transfer between NADP and FAD without electron leakage to other acceptors.

  • Conservation of the Enzyme-Coenzyme Interfaces in FAD and NADP Binding Adrenodoxin Reductase-A Ubiquitous Enzyme.
    Journal of molecular evolution, 2017
    Co-Authors: Israel Hanukoglu
    Abstract:

    FAD and NAD(P) together represent an ideal pair for coupled redox reactions in their capacity to accept two electrons and their redox potentials. Enzymes that bind both NAD(P) and FAD represent large superfamilies that fulfill essential roles in numerous metabolic pathways. Adrenodoxin Reductase (AdxR) shares Rossmann fold features with some of these superfamilies but remains in a group of its own in the absence of sequence homology. This article documents the phylogenetic distribution of AdxR by examining whole genome databases for Metazoa, Plantae, Fungi, and Protista, and determines the conserved structural features of AdxR. Scanning these databases showed that most organisms have a single gene coding for an AdxR ortholog. The sequence identity between AdxR orthologs is correlated with the phylogenetic distance among metazoan species. The NADP binding site of all AdxR orthologs showed a modified Rossmann fold motif with a GxGxxA consensus instead of the classical GxGxxG at the edge of the first βα-fold. To examine the hypothesis that enzyme-coenzyme interfaces represent the conserved regions of AdxR, the residues interfacing FAD and NADP were identified and compared with multiple-sequence alignment results. Most conserved residues were indeed found at sites that surround the interfacing residues between the enzyme and the two coenzymes. In contrast to proteinprotein interaction hot-spots that may appear in isolated patches, in AdxR the conserved regions show strict preservation of the overall structure. This structure maintains the precise positioning of the two coenzymes for optimal electron transfer between NADP and FAD without electron leakage to other acceptors.

  • The structure of Adrenodoxin Reductase of mitochondrial P450 systems: electron transfer for steroid biosynthesis.
    Journal of molecular biology, 1999
    Co-Authors: Gabriele A. Ziegler, Clemens Vonrhein, Israel Hanukoglu, Georg E. Schulz
    Abstract:

    Adrenodoxin Reductase is a monomeric 51 kDa flavoenzyme that is involved in the biosynthesis of all steroid hormones. The structure of the native bovine enzyme was determined at 2.8 A resolution, and the structure of the respective recombinant enzyme at 1.7 A resolution. Adrenodoxin Reductase receives a two-electron package from NADPH and converts it to two single electrons that are transferred via Adrenodoxin to all mitochondrial cytochromesP 450. The structure suggests how the observed flavin semiquinone is stabilized. A striking feature is the asymmetric charge distribution, which most likely controls the approach of the electron carrier Adrenodoxin. A model for the interaction is proposed. Adrenodoxin Reductase shows clear sequence homology to half a dozen proteins identified in genome analysis projects, but neither sequence nor structural homology to established, functionally related electron transferases. Yet, the structure revealed a relationship to the disulfide oxidoReductases, permitting the assignment of the NADP-binding site.

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

  • Raman and infrared spectroscopic evidence for the structural changes of the 2Fe2S cluster and its environment during the interaction of Adrenodoxin and Adrenodoxin Reductase
    Spectrochimica acta. Part A Molecular and biomolecular spectroscopy, 2017
    Co-Authors: Mireille Khalil, Rita Bernhardt, Petra Hellwig
    Abstract:

    Many biological functions involve the formation of proteinprotein complexes. In the present study, we investigated the interaction of two proteins involved in electron transfer, Adrenodoxin (Adx) and Adrenodoxin Reductase (AdR) by using Raman and infrared spectroscopies. Different shifts and splittings of the FeSb/t stretching vibrational modes upon interaction of the two proteins can be reported pointing towards major structural changes in the [2Fe2S] cluster. These changes may be necessary for optimizing electron transfer. The assignment of the shifted modes to the [2Fe2S] cluster was confirmed by 54Fe labeling of the truncated Adx (4-108) as well as the investigation of mutants close to the interaction site and in the vicinity of the [2Fe2S] cluster. Electrochemically induced FTIR difference spectra revealed that the flavin cofactor in AdR also changes due to the interaction with [2Fe2S] cluster in the Adx/AdR electron transfer complex.

  • 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, Rita Bernhardt, Burkhard Schiffler, 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 adenadenineudinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutamutagenesis 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.

  • The interaction domain of the redox protein Adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor Adrenodoxin Reductase.
    Biochemical and biophysical research communications, 2005
    Co-Authors: Achim Heinz, Udo Heinemann, Jurgen Muller, Frank Hannemann, Rita Bernhardt
    Abstract:

    Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in electron transfer reactions in the steroid hormone biosynthesis of mammals. In this study, we deleted the sequence coding for the complete interaction domain in the Adx cDNA. The expressed recombinant protein consists of the amino acids 1-60, followed by the residues 89-128, and represents only the core domain of Adx (Adx-cd) but still incorporates the [2Fe-2S] cluster. Adx-cd accepts electrons from its natural redox partner, Adrenodoxin Reductase (AdR), and forms an individual complex with this NADPH-dependent flavoprotein. In contrast, formation of a complex with the natural electron acceptor, CYP11A1, as well as electron transfer to this steroid hydroxylase is prevented. By an electrostatic and van der Waals energy minimization procedure, complexes between AdR and Adx-cd have been proposed which have binding areas different from the native complex. Electron transport remains possible, despite longer electron transfer pathways.

Georg E. Schulz – One of the best experts on this subject based on the ideXlab platform.

  • Crystal Structures of Adrenodoxin Reductase in Complex with Nadp+ and Nadph Suggesting a Mechanism for the Electron Transfer of an Enzyme Family
    Biochemistry, 2000
    Co-Authors: Gabriele A. Ziegler, Georg E. Schulz
    Abstract:

    Adrenodoxin Reductase is a flavoenzyme that shuffles electrons for the biosynthesis of steroids. Its chain topology belongs to the glutathione Reductase family of disulfide oxidoReductases, all of which bind FAD at equivalent positions. The three reported structures of Adrenodoxin Reductase were ligated with reduced and oxidized NADP and have now confirmed this equivalence also for the NADP-binding site. Remarkably, the conformations and relative positions of the prosthetic group FAD and the cofactor NADP have been conserved during protein evolution despite very substantial changes in the polypeptide. The ligated enzymes showed small changes in the domain positions. When compared with the structure of the NADP-free enzyme, these positions correspond to several states of the domain motion during NADP binding. On the basis of the observed structures, we suggest an enzymatic mechanism for the subdivision of the received two-electron package into the two single electrons transferred to the carrier protprotein Adrenodoxin. The data banks contain 10 sequences that are closely related to bovine Adrenodoxin Reductase. Most of them code for gene products with unknown functions. Within this family, the crucial residues of Adrenodoxin Reductase are strictly conserved. Moreover, the putative docking site of the carrier is rather well conserved. Five of the family members were assigned names related to ferredoxin:NADP(+) Reductase, presumably because Adrenodoxin Reductase was considered a member of this functionally similar family. Since this is not the case, the data bank entries should be corrected.

  • The structure of Adrenodoxin Reductase of mitochondrial P450 systems: electron transfer for steroid biosynthesis.
    Journal of molecular biology, 1999
    Co-Authors: Gabriele A. Ziegler, Clemens Vonrhein, Israel Hanukoglu, Georg E. Schulz
    Abstract:

    Adrenodoxin Reductase is a monomeric 51 kDa flavoenzyme that is involved in the biosynthesis of all steroid hormones. The structure of the native bovine enzyme was determined at 2.8 A resolution, and the structure of the respective recombinant enzyme at 1.7 A resolution. Adrenodoxin Reductase receives a two-electron package from NADPH and converts it to two single electrons that are transferred via Adrenodoxin to all mitochondrial cytochromesP 450. The structure suggests how the observed flavin semiquinone is stabilized. A striking feature is the asymmetric charge distribution, which most likely controls the approach of the electron carrier Adrenodoxin. A model for the interaction is proposed. Adrenodoxin Reductase shows clear sequence homology to half a dozen proteins identified in genome analysis projects, but neither sequence nor structural homology to established, functionally related electron transferases. Yet, the structure revealed a relationship to the disulfide oxidoReductases, permitting the assignment of the NADP-binding site.

  • Chaperone-assisted expression of authentic bovine Adrenodoxin Reductase in Escherichia coli.
    FEBS letters, 1999
    Co-Authors: Clemens Vonrhein, Gabriele A. Ziegler, Israel Hanukoglu, Ulrich Schmidt, Susann Schweiger, Georg E. Schulz
    Abstract:

    Adrenodoxin Reductase is an essential component of the mitochondrial monooxygenase systems that are involved in the synthesis of steroid hormones and related compounds. After removing by mutagenesis a secondary ribosome binding site and an mRNA loop formed between the gene and the vector, large amounts of the enzyme could be produced in Escherichia coli by coexpression with the HSP60-chaperone system. The purified protein was homogeneous enough for reproducible crystallization. The crystals diffracted X-rays isotropically beyond 1.7 A resolution permitting a structure analysis.

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

  • The interaction domain of the redox protein Adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor Adrenodoxin Reductase.
    Biochemical and biophysical research communications, 2005
    Co-Authors: Achim Heinz, Udo Heinemann, Jurgen Muller, Frank Hannemann, Rita Bernhardt
    Abstract:

    Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in electron transfer reactions in the steroid hormone biosynthesis of mammals. In this study, we deleted the sequence coding for the complete interaction domain in the Adx cDNA. The expressed recombinant protein consists of the amino acids 1-60, followed by the residues 89-128, and represents only the core domain of Adx (Adx-cd) but still incorporates the [2Fe-2S] cluster. Adx-cd accepts electrons from its natural redox partner, Adrenodoxin Reductase (AdR), and forms an individual complex with this NADPH-dependent flavoprotein. In contrast, formation of a complex with the natural electron acceptor, CYP11A1, as well as electron transfer to this steroid hydroxylase is prevented. By an electrostatic and van der Waals energy minimization procedure, complexes between AdR and Adx-cd have been proposed which have binding areas different from the native complex. Electron transport remains possible, despite longer electron transfer pathways.

  • covalently crosslinked complexes of bovine Adrenodoxin with Adrenodoxin Reductase and cytochrome p450scc
    FEBS Journal, 2001
    Co-Authors: Evachristina Muller, Anna Lapko, Albrecht Otto, Jurgen Muller, 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, Anna Lapko, Albrecht Otto, Jurgen Muller, 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.

Bruno Dumas – One of the best experts on this subject based on the ideXlab platform.

  • Construction and characterization of a catalytic fusion protein system: P-45011β-Adrenodoxin ReductaseAdrenodoxin
    Biochimica et biophysica acta, 2000
    Co-Authors: Peirang Cao, Bruno Dumas, Hannes E. Bülow, Rita Bernhardt
    Abstract:

    Cortisol is an important intermediate for the production of steroidal drugs and can only be synthesized chemically by rather complicated multi-step procedures. The most critical step is the 11beta-hydroxylation of 11-deoxycortisol, which is catalyzed by a mitochondrial enzyme, P-450(11beta). Various fusion constructs of P-450(11beta) with its electron transfer components, Adrenodoxin and Adrenodoxin Reductase, were produced by cDNA manipulation and were successfully expressed in COS-1 cells from which the hydroxylation activities were assayed. It was demonstrated that the fusion protein required both Adrenodoxin Reductase and Adrenodoxin for its activity and was not able to receive electrons from an external source. The fusion protein with all three components had less activity than P-450(11beta) alone, receiving electrons from coexpressed or internal electron transfer components. The activities of the fusion proteins were determined mainly by the fusion sequence. The fusion protein with a sequence of P-450(11beta)-Adrenodoxin ReductaseAdrenodoxin was more active than that of P-450(11beta)-AdrenodoxinAdrenodoxin Reductase, 1.5- and 3-fold for bovine and human P-450(11beta), respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11beta)-Adrenodoxin ReductaseAdrenodoxin fusion protein indicated that the linker did not have significant effect on the P-450 activity. Taken together, the fusion protein obtained here can serve as a model for the investigation of electron transfer in P-450 systems and is of potential importance for biotechnological steroid production.

  • Characterization of recombinant Adrenodoxin Reductase homologue (Arh1p) from yeast. Implication in in vitro cytochrome p45011beta monooxygenase system.
    The Journal of biological chemistry, 1998
    Co-Authors: Thierry Lacour, Tilman Achstetter, Bruno Dumas
    Abstract:

    The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely Adrenodoxin Reductase and Adrenodoxin. Adrenodoxin Reductase transfers electrons from NADPH to Adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian Adrenodoxin Reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11beta-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent Km values were determined to be 0.5, 0.6, and 0.1 microM for NADPH, NADH, and bovine Adrenodoxin, respectively. These values differ slightly from those of mammalian Adrenodoxin Reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.

  • Characterization of Recombinant Adrenodoxin Reductase Homologue (Arh1p) from Yeast
    , 1998
    Co-Authors: Thierry Lacour, Tilman Achstetter, Bruno Dumas
    Abstract:

    The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely Adrenodoxin Reductase and Adrenodoxin. Adrenodoxin Reductase transfers electrons from NADPH to Adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian Adrenodoxin Reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11b-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent Km values were determined to be 0.5, 0.6, and 0.1 mM for NADPH, NADH, and bovine Adrenodoxin, respectively. These values differ slightly from those of mammalian Adrenodoxin Reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.