Wolfhart Rüdiger

An optomechanical transducer in the blue light receptor phototropin from Avena sativa

The PHOT1 (NPH1) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine/threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13C/15N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1H and 31P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of NPH1.

On the structure of pterobilin, the blue pigment ofPieris brassicae

Aus dem Tegument von Raupen des Kohlweisslings (Pieris brassicae) wird das bereits früher beschriebene Pterobilin isoliert, das auf Grund seiner Abbauprodukte und der chromatographischen und spektralen Eigenschaften als Biliverdin-IXγ identifiziert wird.

Cloning and Characterisation of Chlorophyll Synthase from Avena sativa

Abstract The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in darkgrown and in lightgrown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and Nphenylmaleimide (NPM) inhibit the enzyme activity. Sitedirected mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wildtype and all other Cysmutants with the exception of the mutant C304A are inhibited by Nphenylmaleimide, we conclude that the inhibitor binds to a nonessential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed.

On the biosynthesis of biliverdin-IXγ inPieris brassicae

Radioaktiv markiertes Glyzin wird von Raupen des Kohlweisslings (Pieris brassicae) in Biliverdin-IXγ (Pterobilin), nicht jedoch in die gleichfalls aus dem Tegument isolierten Phäophorbide eingebaut. Die Aktivitätsverteilung in den Abbauprodukten IIIb und IV zeigt, dass dieser Einbau entsprechend dem «normalen» Biosyntheseweg für Gallenfarbstoffe erfolgt.

Various Metallopheophorbides as Substrates for Chlorophyll Synthetase

Abstract Pheophorbide a was prepared from a mixture of chlorophylls a and b by differential extraction with HCl and saponification. The insertion of the following metal ions was investigated: Mg, Zn, Co, Cu, Ni. In the enzyme test with chlorophyll synthetase, the metallopheophorbides fall into two categories: the Mg- and Zn-complexes are good substrates, the Co-, Cu- and Ni-complexes are neither substrates nor competitive inhibitors for the enzyme reaction. This corresponds to two categories of complex structures: Mg- and Zn-porphyrins prefer pentacoordinate square-pyramidal structures, Co-, Cu- and Ni-porphyrins prefer tetracoordinate square-planar structures. A model for substrate binding to chlorophyll synthetase is proposed.

Gallenfarbstoffe bei wirbellosen Tieren

Gallenfarbstoffe (I) entstehen aus Metallkomplexen yon Porphyrinen durch oxydative Ring6ffnung. Die Gallenfarbstoffe der Sdugetiere leiten sich von Protoh~im IX ab (II a), welches spezifisch an der ct-MethinBrticke oxydiert wird: Die entstehenden Biline besitzen die IXa-Struktur 1 [t--3]. Prim~ir bildet sich Bitiverdin (III), alle anderen Biline sind Seknnd~irprodukte [5 ]. Ftir die Ring6ffnung wird entweder ein 16sliches Enzym [6, 7], dessen Existenz aber von einigen Autoren angezweifelt wird [8--t0], oder ein strukturgebundenes Enzymsystem It 1 ] angenommen.

Reduction of the Formyl Group of Zinc Pheophorbide b in vitro and in vivo: a Model for the Chlorophyll b to a Transformation

Abstract The chemical reduction of the formyl group of pheophorbide b with sodium cyanoborohy dride in methanol leads to 71-methoxy-and 71-hydroxy-pheophorbide a. The same reaction with zinc pheophorbide b yields in addition zinc pheophorbide a. This was characterized by mass and 1H -NMR spectroscopy. Infiltration of zinc pheophorbides a and b and of zinc 71-hydroxy-pheophorbide a into etiolated oat leaves yielded phytylated products. The best yield in the esterification was obtained with 71-hydroxy-pheophorbide a. Analysis of the products revealed the formation of zinc pheophytin a from all infiltrated compounds. The significance for the transformation of chlorophyll b into chlorophyll a is discussed.

Preliminary report on the neopterobilins, blue-green pigments from lepidoptera

Abstract 1. 1. A preliminary report on the neopterobilins, blue-green bile pigments from Lepidoptera, is presented. They behave as oxidation products of pterobilin (biliverdin IX γ). 2. 2. Anthereabilin (from the larvae of Antherea pernyi, Attacidae) has the properties of a biladiene ab or bc. 3. 3. Sarpedobilin (from Papilio sarpedon, Papilionidae) could be an hydroxylated bilatriene. 4. 4. Five phorcabilins have been isolated from males of Papilio phorcas; one of them is identical to sarpedobilin; another has been identified with pterobilin.

Zur Bindung von Biliverdin an das Protein im Crenilabrus-Blau

The blue biliprotein from the fins of the Mediterranean fishCrenilabrus pavo C.V. was analysed with respect to linkages between colouring matter (biliverdin IXα) and apoprotein. It was shown by chromic oxidation under various conditions and determination of degradation products that one of the outer and one of the inner rings of biliverdin are free. The remaining inner ring is bound to the apoprotein presumably by an ester bond, while the lability of the linkage between one outer ring and apoprotein corresponds to a N-acyl-bond.

Magnesium chelatase of Hordeum vulgare L. is not activated by light but inhibited by pheophorbide

Abstract The enzyme activity of magnesium chelatase was determined in intact etioplasts of barley (Hordeum vulgare L.) seedlings. Irradiation of isolated plastids with white light for 15 min does not lead to any activation of the enzyme but to a decrease in activity, especially in etioplasts. The enzyme was inhibited by chlorophyllide and zinc pheophorbide only to a certain extent. Strong inhibition was observed with the metal-free pheophorbide (Ki = 0.92 μM) but not with pheophytin or chlorophyll. Penetration of chlorophyllide through the envelope membrane was confirmed by the chlorophyll synthase reaction that occurs in the inner membranes of etioplasts and chloroplasts. The possible role of inhibition of magnesium chelatase by pheophorbide in senescent leaves and tetrapyrrole transport across the plastid envelope are discussed.