The reaction requires reduction equivalents, which in most cases are transferred from a primary electron donor [usually NAD P H] to the actual P enzyme via an electron transport chain consisting of one or two auxiliary proteins. With regard to the composition of the electron transport system, the P systems are grouped into different classes Hannemann et al. Class I comprises most bacterial cytochrome P systems as well as the eukaryotic mitochondrial P systems.
In these systems, the electrons necessary for the monooxygenase reaction are provided by NAD P H via a flavin adenine dinucleotide FAD -containing ferredoxin reductase, which transfers them to a ferredoxin, which in turn reduces the cytochrome P enzyme. In mammals, cytochromes P of the mitochondrial type are essential for the biosynthesis of vitamin D as well as bile acids and of cholesterol-derived steroid hormones.
All these adrenal steroid hydroxylases are provided with reduction equivalents via a short electron transport system consisting of the proteins adrenodoxin reductase AdR, EC 1.
As recently reviewed, the great number of different reactions catalyzed by cytochromes P has attracted not just the attention of academic researchers Bernhardt, ; Munro et al. From an industrial point of view, the catalytic versatility of these enzymes makes their bioproduction a primary challenge to bioscience. In the past years, the fission yeast Schizosaccharomyces pombe emerged as the biotechnologically most attractive host organism for the expression of mammalian cytochromes P Additionally, Schizosaccharomyces pombe can be applied for the production of compounds difficult to synthesize chemically.
For example, genetically engineered Schizosaccharomyces pombe strains have been developed expressing functionally active human cytochromes P CYP11B1 steroid 11b-hydroxylase Dragan et al.
Apart from this, Schizosaccharomyces pombe is capable of serving as an inhibitor test organism for recombinant human steroid hydroxylases CYP17, CYP21 Dragan et al. All these cytochrome Pbased applications in fission yeast rely on a functionally active electron transport chain to the heterologously expressed cytochromes P But despite the dependence on auxiliary proteins, it has been reported previously that Schizosaccharomyces pombe cells expressing human CYP11B2 display in vivo activity without coexpression of the natural redox partner proteins Bureik et al.
Evidently, the proteins forming this endogenous redox chain play a crucial role in enabling Schizosaccharomyces pombe to provide electrons for mitochondrial cytochrome P enzymes without the demand for coexpression of the natural redox partners. Interestingly, coexpression of adrenodoxin, which is not necessary in fission yeast, is essential to obtain cortisol in the baker's yeast whole cell system Dumas et al. In fission yeast only the endogenous ferredoxin component etp1 fd of the fission yeast redox chain has been described so far and, in addition to in vivo studies, a detailed in vitro analysis of its interaction with several cytochrome Ps has been described Bureik et al.
Overexpression of the endogenous fusion protein etp1, consisting of a ferredoxin domain and a domain homologous to COX15 of Saccharomyces cerevisiae , was found to enhance P activity in CYP11B2 expressing fission yeast cells Bureik et al. The carboxyterminal domain of the fusion protein is cleaved off after import into mitochondria, yielding the adrenodoxin homolog etp1 fd.
The ferredoxin domain was previously expressed in Escherichia coli , and the purified protein was shown to be capable of substituting for adrenodoxin in cytochrome Pdependent substrate conversion assays with bovine CYP11A1 and CYP11B1 Schiffler et al. We report here a detailed bioinformatic analysis as well as a successful cloning, heterologous expression and purification of the putative interaction partner of etp1 fd , named adrenodoxin reductase homolog 1 arh1.
Furthermore, by introducing a mutation into the FAD-binding region leading to a single amino acid exchange, we could obtain a more stable arh1 variant with respect to the incorporation of the prosthetic group FAD, which is easily lost in the wildtype. The spectral characteristics confirm the similarity of the protein with mammalian AdR. The sequence derived from the Schizosaccharomyces pombe genome project Wood et al. The program targetp V1. Protein sequence alignments were done using the alignx function of vector nti advance10 from Invitrogen.
After centrifugation at 20 g for 2 min, the upper aqueous phase was re-extracted with phenol : chloroform : isoamyl alcohol 25 : 24 : 1 and then with chloroform : isoamyl alcohol 49 : 1 for 1 min each. The respective fragments were inserted into the E. The sequence identity of the inserts was verified by DNA sequencing.
Paired oligonucleotides were designed with the program silent site selector. Arh1 was purified via immobilized metal ion affinity chromatography IMAC. The column was then washed with buffer A and the His-tagged target protein eluted with buffer B 50 mM sodium acetate, pH 4. The eluted protein was collected and the buffer exchanged to mM potassium phosphate, pH 7. Later, the sample was concentrated to a final concentration of 0.
Total protein quantification of the purified protein was carried out using the BC Assay kit Uptima following the manufacturer's protocol. The percentage of holoenzyme in the preparation was calculated from the concentration obtained by UV—Vis spectroscopy vs. Recombinant bovine adrenodoxin and AdR were purified as reported previously Uhlmann et al. The preparation of the ferredoxin domain of the fission yeast protein etp1 fd was described previously Bureik et al. In the case of arh1 wildtype and AdR, this treatment resulted in the denaturation of the protein and in the release of the flavin cofactor to the supernatant.
Solvent A was 0. Optical difference spectroscopy was performed based on Warburg's optical test. Simultaneously, an absorbance decrease at nm indicates the formation of reduced arh1. Difference spectroscopy was performed in tandem cuvettes. Identical amounts of reducing agent i. NADPH or NADH were added to the buffer chamber in the reference cuvette and to the reductase solution in the sample cuvette, respectively, and the difference spectra were recorded. The assay was performed in a reconstituted system as described by Sugano with slight alterations.
Samples were extracted with chloroform, dried and dissolved in acetonitrile. The steroid cortisol F was added to the samples as an internal standard in order to correct the obtained substrate conversion values for the loss during extraction.lastsurestart.co.uk/libraries/answer/3608-track-a.php
Analysis of the DNA sequence of this gene revealed the presence of two exons of and bp, respectively. Accordingly, the arh1 cDNA was expected to be bp long. Translation of this DNA sequence theoretically yields a polypeptide of amino acids. Analysis of the primary structure of arh1 led to the identification of a mitochondrial localization signal composed of the first 10 amino-terminal amino acids.
Flavoprotein Protocols — Heriot-Watt University
This prediction corresponds with the report of the ORFeome project in fission yeast Matsuyama et al. The mitochondrial localization of both electron transfer proteins, arh1 and etp1 fd , renders them suitable for donating electrons to mitochondrial cytochromes P Mature arh1 was predicted to have an pI of 9.
The latter is among the values of related proteins such as bovine AdR However, the pI of Schizosaccharomyces pombe arh1 is definitely more similar to that of the Saccharomyces cerevisiae homolog, ARH1 9. In order to further analyze the similarity of arh1 to its homologs, the amino acid sequences of bovine AdR, human AdR, Schizosaccharomyces pombe arh1 and Saccharomyces cerevisiae ARH1 were aligned Fig.
Based on these values, fission yeast arh1 seems to be more similar to the mammalian AdR than to its baker's yeast counterpart. When aligned to bovine and human AdR, it contains Saccharomyces cerevisiae ARH1, on the other hand, shows only an identity of Alignments of ferredoxin reductase amino acid sequences.
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Residues are marked as follows: nonsimilar, black on white; conservative, white on dark gray; block of similar, dark gray on light gray; identical, white on black; weakly similar: dark gray on gray. The two Arg residues in AdR, which are mainly responsible for the interaction with adrenodoxin Vickery, , are highlighted by arrow heads and underlined.
Protein sequence identity and similarity values of mature forms of different ferredoxin reductases. Percentage identity is given in bold numbers, percentage similarity in parentheses. To generate the alignment, sequences without mitochondrial signal peptide were used. Considering the alignment depicted in Fig.
Whereas this motif is unaltered in Saccharomyces cerevisiae ARH1, Schizosaccharomyces pombe arh1 differs with the last conserved glycine being replaced by an alanine residue, which might be an explanation for the unusually weak binding of the flavin we observed. Nevertheless, due to its overall high identity to AdR, Schizosaccharomyces pombe arh1 definitely belongs to this family of enzymes. The second highly conserved part shown in Fig. This feature of the mammalian AdR is shared by Schizosaccharomyces pombe arh1. An additional change from alanine to serine in the consensus sequence occurs at the third conserved residue in arh1.
The third conserved area represents one of the two interaction regions important for the interaction of AdR with adrenodoxin. It contains two arginine residues R and R of human AdR , which are supposed to mainly direct the protein—protein interaction Vickery, These residues are absolutely conserved in both mammalian AdRs and Schizosaccharomyces pombe arh1. In addition, the residues on the ferredoxin surface involved in the complex formation are equally well conserved in adrenodoxin and etp1 fd Bureik et al. This suggests a mechanistically similar interaction of the mammalian and Schizosaccharomyces pombe proteins during the electron transfer reaction.
In order to check this hypothesis, expression plasmids were constructed for the heterologous expression of Schizosaccharomyces pombe arh1 in E. The identity of the PCR product was verified by DNA sequencing, which revealed the presence of two silent point mutations. In Fig. The bright yellow color of the lysate reveals abundant amount of holoprotein.
After IMAC purification, a single band of identical size proved the successful purification of the protein. The obtained value of To exclude the possibility that the apparent flavin loss was due to the presence of the His-tag, we also cloned, expressed and purified the mature form of arh1 without any tag. We found that binding of the cofactor in this protein is likewise very weak and the protein lost its flavin cofactor almost completely after applying the second purification step, irrespective of the applied chromatography type data not shown.
From this result we assumed that the variation of the FAD-binding motif from GxGxxG to GxGxxA in Schizosaccharomyces pombe arh1 might be responsible for the unusually weak binding of the flavin cofactor. Consequently, we exchanged the codon for alanine to glycine in position 18 in the His-tagged arh1 construct and found that this single amino acid exchange in fact resulted in a drastically increased yield of holoprotein.
AdR, with local absorption maxima at and nm as well as a shoulder at nm. Moreover, a comparison of the protein concentration determined by the molar extinction coefficient at nm and by a colorimetric assay confirmed that the flavin cofactor is present in the mutant. The inset of Fig. UV-Vis spectra of S. The absorbance maxima of the oxidized protein spectra are at , and nm.
Doerrier 2017 Abstract MitoEAGLE Barcelona
In the inset, the mean values of percent holoenzyme in arh1 wildtype [light gray; In order to confirm the nature of the flavin cofactor — i. For both, the wildtype and the mutant form of arh1, we obtained a single, clear peak with a molecular mass of Because the protein after site-directed mutagenesis was available as a stable holoprotein, the first step of the electron transfer, i.
In both cases, typical signals appeared at and nm, indicating the oxidation of the reducing agent NAD P H and the reduction of arh1. Upon addition of the reducing agent, in both cases a drastic absorbance decrease at nm was observed, which indicates the oxidation of NAD P H. Furthermore, the reduction of the reductase can also be deduced more directly from the decrease appearing at nm, which mirrors the depletion of the typical, flavin-dependent maximum of the oxidized reductase. After having established that arh1 can be reduced by NADPH like its mammalian homolog, we now wanted to investigate the capacity of reduced arh1 to transfer electrons to etp1 fd.
As reported previously, recombinant etp1 fd may be used instead of adrenodoxin in cytochrome P catalyzed steroid conversion assays Schiffler et al. In a reconstitution assay consisting of bovine CYP11A1 and recombinant ferredoxin and ferredoxin reductase, the conversion reaction was compared for all different combinations of the proteins constituting the electron transport chain i. Figure 5a presents an example of two typical HPLC chromatograms recorded after the said steroid conversion reactions.
Reduction of the flavins induces the dissociation of the holocomplex into apododecin and free flavin. Based on these unique binding characteristics, a molecular crane shall be developed that is able to pick up and to release molecular objects through a switch of the electric potential.