Günter F. Wildner

Linkage between Catecholate Siderophores and the Multicopper Oxidase CueO in Escherichia coli

The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Phi(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.

A new mutation in the gene coding for the herbicide-binding protein in Chlamydomonas

Sequence analysis of herbicide‐resistant Chlamydomonas mutants revealed a new mutation in the psb A gene coding for the herbicide‐binding (D1) protein. A point mutation at codon 251 leading to an amino acid substitution from alanine in wild‐type cells to valine in the mutant was identified by sequencing total cellular RNA. The folding pattern of D1 predicts Ala 251 to be part of the Qb‐binding niche.

Formation of the small subunit in the absence of the large subunit of ribulose 1,5-bisphosphate carboxylase in 70 S ribosome-deficient rye leaves

Abstract Immunological tests with monospecific antisera to ribulosebisphosphate carboxylase (EC 4.1.1.39) and to its large and small subunits indicated the presence of a protein with antigenic properties of the small subunit in the absence of the large subunit in the leaves of young rye plants (Secale cereale L.) with a high-temperature-induced (32 °C) deficiency of 70 S plastid ribosomes. The small subunit-like protein was isolated from crude extracts of plastid ribosome-deficient 32 °C-grown leaf tissue by the use of columns with immobilized antibody. The main polypeptide retained by the immobilized antibodies had the same mobility after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels as the small subunit of ribulosebisphosphate carboxylase and was also immunologically identical to the small subunit. The small subunit-like protein was present in the supernatant as well as in the membrane fraction of isolated 70 S ribosome-deficient plastids. At very young stages of normal leaves grown at a permissive temperature (22 °C) an excess of small subunit was observed that was also not integrated into the complete ribulosebisphosphate carboxylase molecule. From the results, we conclude that the synthesis of the small subunit occurs on cytoplasmic ribosomes and is not strictly coordinated with the translation of the large subunit in the chloroplast. During early leaf development, the formation of the large subunit seems to be the ratelimiting step in the synthesis of ribulosebisphosphate carboxylase.

The effect of divalent metal ions on the activity of Mg(++) depleted ribulose-1,5-bisphosphate oxygenase.

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg++. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO2. Incubation with Mg++ and CO2 restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn++, Co++, and Ni++) and CO2 fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO2-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn++ ions were present. Other divalent cations such as Ca++ and Zn++, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn++ to the Mg++ and CO2 preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.

N-terminal sequence analysis of the 8 kDa protein in Chlamydomonas reinhardii Localization of the phosphothreonine

A phosphorylated 8 kDa protein of Chlamydomonas reinhardii thylakoids has been isolated and its N‐terminal amino acid sequence determined by gas‐phase sequencing. The sequence analysis of the 48 amino acid residues revealed that this protein is about 50% homologous to the psb H gene products of higher plants. In contrast to them, it contains an insert of seven amino acid residues (Ser‐5 to Lys‐11). The first threonine residue was phosphorylated as determined by 32P detection during sequencing and also by analysis of the modified degradation products in the chemical reaction of the Edman degradation process. This latter method allows the identification of phosphorylated threonine residues without radiolabelling the protein.

Differential reactivation of ribulose 1,5-bisphosphate oxygenase with low carboxylase activity by Mn2+

Ribulose 1 ,5-bisphosphate carboxylase-oxygenase (EC 4.1 .1.39) catalyses either the carboxylation or the oxidative splitting of ribulose 1,5_bisphosphate (RuBP) [l]. Both enzyme activities are stimulated by incubation with Mg2’ and bicarbonate, both of which are necessary in converting the enzyme molecules to an active enzyme-COa-Mg’+ complex [2-S]. Kinetic analysis [6] suggested that the activating sites of the enzyme molecules are not identical with the substrate binding sites for COa. The role of divalent cations is not confined to the formation of this ternary complex, since divalent cations are also bound [7] to the second substrate RuBP at the active site of the enzyme. Mn*+ can substitute for Mg*+ in the formation of a quaternary complex, enzyme-RuBP-Me*‘-COa, with RuBP in the inner sphere and CO2 in the outer sphere of enzyme bound Mn’+. The studies concerning the replacement of Mg*+ by Mn2+ and its influence on the carboxylase activity showed diverging results, in some experiments the presence of Mn2+ could not activate the enzyme [g-lo], whereas other data supported a stimulatory role for this ion t111. The aim of this paper is to describe the influence of Mn2+ on the RuBP oxygenase and to reevaluate the discrepancies on the carboxylase function. The reconstitution experiments revealed that MnZC could replace Mg2* ions thereby fully restoring the oxygenase activity; however, the carboxylase activity was only slightly enhanced. 2.1. Enzyme purification RuBP carboxylase-oxygenase was isolated from spinach leaves. The purification process including ammonium sulfate precipitation (30-55% saturation), Sephadex G-200 gel filtration and ultracentrifugation on a sucrose step gradient were detailed in [ 121. The enzyme fraction with the highest specific activity (1.2 U/mg protein) showed a single band in 5% acrylamide gels after disc electrophoresis [ 131.

Localization of the reaction site of cytochrome 552 in chloroplasts from Euglena gracilis: Cytochrome content and photooxidation in different chloroplast preparations☆

Abstract Isolated Euglena chloroplasts retain up to 50% of cytochrome 552 on a chlorophyll basis compared to the content of cells. Cytochrome 563 is found in equal amount in chloroplasts and cells. The amount of cytochrome 552 retained depends on the isolation procedure of chloroplasts. Cytochrome 552 can be further liberated from chloroplasts by mechanical treatment or incubation with detergent. It is concluded that cytochrome 552 is not tightly bound in the membrane but rather trapped in the thylakoids of the chloroplasts. In photosynthetic electron flow, cytochrome 552 is functioning as donor for photosystem I, mediating electron flow from cytochrome 558 to P 700 under our conditions. Antimycin A stimulates the photooxidation of cytochrome 552 and of cytochrome 558. The rates of electron flow from water to NADP + and of cyclic photophosphorylation mediated by phenazine methosulfate correlate with the content of endogenous cytochrome 552 in the chloroplasts. External readdition of cytochrome 552 to deficient chloroplasts causes reconstitution of NADP + reduction but not of cyclic photophosphorylation. Mechanical treatment or other means of fragmentation of chloroplasts results in the exposure of originally buried reaction sites for external cytochrome 552.

Localization of the reaction site of cytochrome 552 in chloroplasts from Euglena gracilis. Effects of a specific antibody on endogenous, external and reincorporated cytochrome 552 in chloroplast membranes.

Abstract Euglena chloroplasts, isolated by Yeda press treatment contain endogenous cytochrome 552. Antibodies against cytochrome 552 from Euglena gracilis do not agglutinate chloroplasts and do not inhibit photosynthetic electron flow from water to NADP + . There is also no influence on cyclic photophosphorylation with phenazine methosulfate as mediator and on photooxidation of endogenous cytochrome 552. However, in the presence of cholate the photooxidation of the cytochrome is inhibited by antibodies. Cyclic photophosphorylation is not restored by addition of cytochrome 552 to the assay mixture but is stimulated by trapping the cytochrome in the thylakoid vesicles during sonication. Trapped cytochrome 552 is not accessible to antibodies. It is concluded that the original site of action for endogenous cytochrome 552 is inside the thylakoids. This site can be dislocated to the outside during fragmentation of chloroplasts.

Formation of the tight-binding inhibitor, 3-ketoarabinitol-1,5-bisphosphate by ribulose-1,5-bisphosphate carboxylase/oxygenase is O2-dependent

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) not only catalyzes carboxylation and oxygenation of ribulose-1,5-bisphosphate (RuBP), but it can also act either as an epimerase or isomerase converting RuBP into xylulose-1,5-bisphosphate (XuBP) or 3-ketoarabinitol-1,5-bisphosphate (KABP), respectively, a process called misfire. XuBP is formed as a result of misprotonation at C3 of the RuBP-enediol. It is released from Rubisco active sites and accumulates in the reaction mixture. Increasing the amounts of CO2 or O2 decreases XuBP production. However, KABP synthesis, which has been proposed to be only a product due to C2 misprotonation of the RuBP-endiol, is dependent upon the presence of O2. KABP remains tightly bound to Rubisco active sites after its formation, causing the loss of Rubisco activity (‘fallover’). The results suggest that the non-stabilized form of the peroxy-intermediate in the oxygenase reaction can be converted in a backreaction to KABP and molecular oxygen. The stabilization of the peroxy-intermediate due to the presence of Mn2+ instead of Mg2+ eliminates the formation of KABP.

Specific inhibition of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase

Summary Ribulose-1,5-bisphosphate oxygenase activity of ribulose-1,5-bisphosphate carboxylase was inhibited 82% by incubation with 5 mM glycidate (2,3-epoxyproprionate). Under these conditions the carboxylase was not affected. Similar results have also been obtained with the S-alkylating agent, iodoacetamide. These observations suggest the involvement of sulfhydryl-groups in the ribulose-1,5-bisphosphate oxygenase catalysis. Furthermore, these results may explain the role of glycidate in the inhibition of glycolate synthesis and the net increase of photosynthesis in tobacco leaf discs as demonstrated by Zelitch (1).