Florence K. Gleason

The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase

The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a beta-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.

Site of action of the natural algicide, cyanobacterin, in the blue-green alga, Synechococcus sp.

Cyanobacterin is a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni. Highly purified cyanobacterin was found to inhibit the growth of many cyanobacteria at a minimum effective dose of 2 μg/ml (4.6 μM). The antibiotic had no effect on eubacteria including the photosynthetic Rhodospirillum rubrum. The site of action of cyanobacterin was further investigated in the unicellular cyanobacterium, Synechococcus sp. Electron micrographs of antibiotic-treated Synechococcus cells indicated that cyanobacterin affects thylakoid membrane structure. The antibiotic also inhibited light-dependent oxygen evolution in Synechococcus cells and in spheroplasts. These data support our conclusion that cyanobacterin specifically inhibits photosynthetic electron transport. This activity is similar to herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). The anhydro analog of cyanobacterin had no biological activity.

Crystal structure of thioredoxin-2 from Anabaena.

BACKGROUND Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases. The structures of thioredoxins from a number of species, including man and Escherichia coli, are known. Cyanobacteria, such as Anabaena, contain two thioredoxins that exhibit very different activities with target enzymes and share little sequence similarity. Thioredoxin-2 (Trx-2) from Anabaena resembles chloroplast type-f thioredoxin in its activities and the two proteins may be evolutionarily related. We have undertaken structural studies of Trx-2 in order to gain insights into the structure/function relationships of thioredoxins. RESULTS Anabaena Trx-2, like E. coli thioredoxin, consists of a five-stranded beta sheet core surrounded by four alpha helices. The active site includes a conserved disulfide ring (in the sequence 31WCGPC35). An aspartate (E. coli) to tyrosine (Trx-2) substitution alters the position of this disulfide ring relative to the central pleated sheet. However, loss of this conserved aspartate does not affect the disulfide geometry. In the Trx-2 crystals, the N-terminal residues make extensive contacts with a symmetry-related molecule with hydrogen bonds to residues 74-76 mimicking thioredoxin-protein interactions. CONCLUSIONS The overall three-dimensional structure of Trx-2 is similar to E. coli thioredoxin and other related disulfide oxido-reductases. Single amino acid substitutions around the protein interaction area probably account for the unusual enzymatic activities of Trx-2 and its ability to discriminate between substrate and non-substrate peptides.

The role of calcium in the regulation of free radical reduction by thioredoxin reductase at the surface of the skin

Membrane associated thioredoxin reductase has been previously shown to reduce free radicals on the outer plasma membranes of human keratinocytes and melanocytes to provide a possible first line of defense against free radical damage at the surface of the skin. Preliminary experiments with cell cultures of human keratinocytes and melanocytes grown in serum-free medium showed that the enzyme activity depends on extracellular calcium concentration in the medium. Thioredoxin reductase activity at the surface of the skin, at the surface of human keratinocytes and melanocytes, and purified thioredoxin reductase from E. coli and adult human keratinocytes all exhibited calcium-dependent allosteric control. Since thioredoxin reductase contains two extremely reactive thiolate groups at the active site with pK values close to neutrality, both of these anions can form covalent complexes with N-ethylmaleimide by nucleophilic attack on the double bond. In our experiments we used spin-labeled maleimide [4-maleimido-tempo] to examine the local environment in the active site of thioredoxin reductase in the presence and absence of calcium. Both spin-labeled thioethers are distinguishable by EPR spectroscopy, with one site being significantly more immobilized than the other. Hence, it has been possible to observe direct evidence for active site closure by calcium. These results suggest that extracellular calcium may play an important role in regulation of thioredoxin reductase activity for the defense mechanism against UV-mediated free radical damage at the surface of human skin.

Effect of the natural algicide, cyanobacterin, on a herbicide-resistant mutant of anacystis nidulans R2

Abstract Cyanobacterin, a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni , inhibits the growth of algae and plants. This compound is a potent inhibitor of photosynthetic electron transport and acts at a site in photosystem II (PS II). To further define the site of action of cyanobacterin, the effects of this natural product were investigated in a herbicide-resistant mutant of the cyanobacterium, Anacystis nidulans R2D2-X1. A. nidulans R2D2-X1 was reported to grow and maintain photosynthetic electron transport in the presence of 20 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 6.0 μM atrazine. Resistance was attributed to an altered 32 kDa (quinone-binding, QB) protein [6]. In the presence of Hill electron acceptors, K 3 Fe(CN) 6 and dichlorophenol-indophenol (DCPIP), spheroplasts of A. nidulans R2D2-X1 were inhibited by cyanobacterin at the same concentration as wild type spheroblasts. Under these same conditions, spheroplasts of the mutant maintained their resistance to DCMU. Similar results were obtained with isolated thylakoid membranes. In contrast, silicomolybdate reduction, which is resistant to DCMU inhibition, was very sensitive to cyanobacterin. We conclude that cyanobacterin inhibits electron transport in PS II at a unique site which is different from that of DCMU.

A second algicidal natural product from the cyanobacterium, Scytonema hofmanni

Abstract We had previously reported the isolation and characterization of an herbicidal metabolite, cyanobacterin, from the freshwater cyanobacterium (cyanophyte), Scytonema hofmanni UTEX 2349. This compound was identified as a chlorine-containing, diaryl-substituted, γ-lactone. The natural product inhibits electron transport in photosystem II and is toxic to a number of algae and higher plants. Crude cell extracts of Scytonema contain other chlorinated metabolites with biological activity. We have isolated and characterized a more hydrophilic analog of cyanobacterin in which an hydroxyl group replaces the methylene dioxy group on the chlorine-containing aromatic ring. Although it is an effective inhibitor of photosynthetic electron transport in isolated thylakoid membranes, the analog has little effect on the growth of intact algal cells. It readily dehydrates in air at 25°C. The hydroxy analog is present in very small quantities in Scytonema cells and is probably a metabolic precursor of cyanobacterin.

Characterization of a mutant of Anacystis nidulans r2 resistant to the natural herbicide, cyanobacterin

Abstract Cyanobacterin, a secondary metabolite produced by the cysnobacterium, Scytonema hofmanni, inhibits electron transport at a site in photosystem II. It was previously shown that a DCMU-resistant mutant of A. nidulans R2 was still susceptible to cyanobacterin (Gleason et al., Plant Science, 46 (1986) 5–10). Apparently, cyanobacterin acts at a site different from that of DCMU and similar PS II inhibitors. To confirm this conclusion, a cyanobacterin-resistant strain of A. nidulans R2 was produced by nitrosoguanidine mutagenesis and selected by growth in the presence of 4.7 μM cyanobacterin. Hill activity in mutant thylakoids was compared to that of the wild type membranes in the presence of ferricyanide and silicomolybdate as electron acceptors. Photosynthetic electron transport in the mutant membranes shows a high degree of resistance to cyanobacterin in both reactions. In contrast, the mutant exhibits the same susceptibility to DCMU inhibition as the wild type R2. Cyanobacterin acts at a unique site, inhibiting electron flow from quinone-A to quinone-B.

Thioredoxins in Cyanobacteria: Structure and Redox Regulation of Enzyme Activity

Thioredoxin is a small disulfide-containing redox protein that reduces disulfide bonds in other proteins. It can act both as a modulator of enzyme activity by reducing structurally important disulfide bonds in a target protein and also as a reducing agent. Unlike other eubacteria, the cyanobacteria have two distinct thioredoxins with approximately 39% amino acid identity. One of the thioredoxins (T1) is similar to bacterial thioredoxins both in structure and general redox activity. The other protein (T2) is relatively unstable and seems to be unique to cyanobacteria. Several enzymes in cyanobacteria are regulated by a disulfide redox mechanism that can be effected by either T1 or T2, including enzymes for CO2 fixation, carbon catabolism and nitrogen metabolism. In addition, cyanobacterial thioredoxins can function as reducing agents in the phosphoadenosine phosphosulfate reductase and ribonucleotide reductase reactions. Thioredoxins, in turn, are reduced by the products of PS I, thus providing a biochemical link between light reactions and regulation of metabolism. An attempt to inactivate the Tl gene in Synechococcus sp. strain PCC 7942 did not succeed in producing thioredoxin-minus mutants which implies that T1 performs some essential function in photosynthetic organisms that cannot be efficiently substituted by T2 or other redox systems. Although its role in regulating carbon metabolism seems to be most crucial for photoautotrophic growth, definitive evidence for the in vivo functions of thioredoxin in cyanobacteria is still lacking.

Control of ribonucleotide reductase in Synechococcus Sp., a unicellular cyanobacterium

Cells of Synechococcus sp., a rod-shaped, unicellular cyanobacterium (blue-green alga) can be readily snychronized by depriving the cells of carbon dioxide and light for a 12 h period. On resumption of growth, a portion of the population undergoes two sharply synchronized divisions. Ribonucleotide reductase activity was found to be maximal during the time of DNA synthesis in these cells. The peak of reductase activity could be abolished by adding inhibitors such a chloramphenicol to the culture, suggesting that the enzyme is induced at the gene level in the cyanobacteria. Additional properties of ribonucleotide reductase were investigated in Synechococcus cells made permeable by treatment with ether. Cytidine triphosphate reduction is absolutely dependent on adenosylcobalamin (coenzyme B12) and is subject to allosteric stimulation by deoxyadenosine triphosphate.

Cyanobacterin and Analogs: Structure and Activity Relationships of a Natural Herbicide

Cyanobacterin is a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni UTEX 2349, which inhibits the growth of other algae. The compound is a diaryl-substituted γ-lactone (Table 1). Cyanobacterin interrupts photosynthetic electron transport at a site in PS II. Analysis of PS II activity with various Hill electron acceptors suggests that the site of action is not identical to the classical PS II electron transport inhibitors (1).