Ribonucleotide reductase in blue-green algae: dependence on adenosylcobalamin.

Ten species of freshwater blue-green algae exhibit an adenosylcobalamin-dependent ribonucleotide reductase, thuse explaining the requirement for cobalt by these organisms. The evidence suggests a phylogenetic affinity between the cyanophytes and bacteria, such as Clostridium and Rhizobium, and the euglenoid flagellates, which also use the cofactor-dependent reductase. In contrast, the ribonucleotide reductase reaction in the few green algae surveyed shows no dependence on cobalamins.

Isolation of chlorine-containing antibiotic from the freshwater cyanobacterium Scytonema hofmanni.

Scytonema hofmanni, a filamentous freshwater cyanobacterium (blue-green alga), produces secondary metabolites which inhibit the growth of other cyanobacteria and green algae. A rapid, qualitative assay for this inhibition has been developed with Synechococcus as the test organism. This assay procedure has led to the isolation and characterization of an antibiotic (named cyanobacterin) from Scytonema. The antibiotic has a molecular weight of 430 and an empirical formula of C23H23O6Cl and contains a gamma-lactone and a chlorinated aromatic nucleus. It inhibits the growth of various algae but has limited effect on nonphotosynthetic bacteria or protozoans and thus may have potential use as a specific algicide.

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.

Thioredoxin and related proteins in procaryotes.

Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)2] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control. Thioredoxin-negative mutants (trxA) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.

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.

Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function.

Mutations were made in three highly conserved residues in Escherichia coli thioredoxin. An internal charged residue, Asp-26, was changed to an alanine. The mutant protein was more stable than the wild type. It can function as a substrate for thioredoxin reductase with a 10-fold increase in the Km over the wild type. Although the redox potential was not substantially changed from that of the wild type, thioredoxin D26A was a poor reducing agent for ribonucleotide reductase. Asp-26 apparently serves to maintain an optimal charge distribution in the active site region for interaction with other proteins. Mutation of a surface Pro-34 in the active site disulfide ring to a serine had little effect on protein stability. A slight decrease in the redox potential (9 mV) made thioredoxin P34S a better reducing agent for ribonucleotide reductase. In contrast, mutation of the internal cis Pro-76 to an alanine destabilized the protein. The data indicate a change had also occurred in the charge distribution in the active site region. Thioredoxin P76A had a higher redox potential than the wild type protein and was not an effective reducing agent for ribonucleotide reductase. It was concluded that this residue is essential for maintaining the conformation of the active site and the redox potential of thioredoxin.

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.

Activities of two dissimilar thioredoxins from the cyanobacterium Anabaena sp. strain PCC 7120.

Thioredoxin is a small redox protein that functions as a reducing agent and modulator of enzyme activity. A gene for an unusual thioredoxin was previously isolated from the cyanobacterium Anabaena sp. strain PCC 7120 and cloned and expressed in Escherichia coli. However, the protein could not be detected in Anabaena cells (J. Alam, S. Curtis, F. K. Gleason, M. Gerami-Nejad, and J. A. Fuchs, J. Bacteriol. 171:162-171, 1989). Polyclonal antibodies to the atypical thioredoxin were prepared, and the protein was detected by Western immunoblotting. It occurs at very low levels in extracts of Anabaena sp. and other cyanobacteria. No antibody cross-reaction was observed in extracts of eukaryotic algae, plants, or eubacteria. The anti-Anabaena thioredoxin antibodies did react with another unusual thioredoxin-glutaredoxin produced by bacteriophage T4. Like the T4 protein and other glutaredoxins, the unusual cyanobacterial thioredoxin can be reduced by glutathione. The Anabaena protein can also activate enzymes of carbon metabolism and has some functional similarity to spinach chloroplast thioredoxin f. However, it shows only 23% amino acid sequence identity to the spinach chloroplast protein and appears to be distantly related to other thioredoxins. The data indicate that cyanobacteria, like plant chloroplasts, have two dissimilar thioredoxins. One is related to the more common protein found in other prokaryotes, and the other is an unusual thioredoxin that can be reduced by glutathione and may function in glucose catabolism.

Glucose-6-phosphate dehydrogenase from the cyanobacterium, Anabaena sp. PCC 7120: purification and kinetics of redox modulation.

Glucose-6-phosphate dehydrogenase is a particularly important enzyme in carbon catabolism in the chloroplasts of higher plants and in cyanobacteria. It catalyzes the first reaction in the oxidative pentose phosphate pathway which supplies reduced NADP for a variety of biosynthetic processes. The enzyme is known to be regulated by light. However, the dehydrogenase from plants has been difficult to purify and there is little information on kinetics and mechanism of deactivation. The glucose-6-phosphate dehydrogenase from the heterocystous cyanobacterium, Anabaena sp. PCC 7120, was purified to near homogeneity by chromatography on 2',5'-ADP Sepharose chromatography. The cyanobacterial enzyme apparently has different aggregation states or conformations depending on its concentration in solution and the pH. At a pH of 8.0 and low ionic strength, the enzyme has relatively low activity and exhibits sigmoidal kinetics on binding substrate and cofactor. Activity increases and the enzyme exhibits the more classical hyperbolic kinetics at pH 7.0. At the lower pH, glucose-6-phosphate dehydrogenase is inhibited by catalytic amounts of reduced thioredoxin-1 from Anabaena sp. The second thioredoxin from the cyanobacterium is much less effective, although its inhibitory effect is still greater than that of small molecule reducing agents such as glutathione. Glutamine was reported to stabilize the isolated enzyme, but actually is an activator at pH 8.0. The results suggest that cellular demand for reduced cofactor under nitrogen-fixing conditions overrides the pH-induced deactivation.

Fatty acids of mitochondrial membranes from Tetrahymena pyriformis.

We have examined the fatty acid composition of the mitochondrial membranes in three strains of Tetrahymena pyriformis. All three had similar components and exhibited large amounts of unsaturated fatty acids. The cytoplasmic mutant, CA-10, which has a slower growth rate and unusual membrane morphology, had a slightly higher amount of iso-acids but was otherwise similar to the other strains in fatty acid composition. Arachidonic acid, previously undetected in extracts of Tetrahymena, was identified as a minor component of the mitochondrial membrane.

Adenosylcobalamin-dependent ribonucleotide reductase from the blue-green alga, Anabaena sp. Purification and partial characterization.

The ribonucleotide reductase from Anabaena 7119 has been purified approximately 60- to 80-fold by conventional techniques and adsorption to the affinity medium, Matrix Gel Red A. The enzyme from Anabaena resembles the adenosylcobalamin-dependent reductase from Lactobacillus leichmannii, in that it is a small molecule (molecular weight 72,000) with no subunit structure as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Unlike its prototype, the Anabaena reductase is absolutely dependent on a divalent cation for activity, Ca2+ being the most effective. In addition, the Anabaena reductase shows a simple pattern of alloteric control by deoxyribonucleotides. CTP reduction is stimulated by dATP, GTP by dTTP, and ATP by dGTP. No reduction is observed in the absence of effectors, and none of the effectors inhibits enzyme activity. Thus, the Anabaena ribonucleotide reductase can be more easily studied by kinetic analysis than the Lactobacillus enzyme, and should provide additional information as to the mechanism of action of this enzyme in a photosynthetic organism.

Isolation and characterization of thioredoxin from the cyanobacterium, Anabaena sp.

Thioredoxin from Anabaena sp. has been purified 800-fold with an assay based on the reduction of insulin disulfides by NADPH and the heterologous calf thymus thioredoxin reductase. The final material was homogeneous on polyacrylamide gel electrophoresis and had a molecular weight of 12,000; the NH2-terminal residue was serine and the COOH-terminal was leucine. Anabaena thioredoxin-(SH)2 is a hydrogen donor for the adenosylcobalamin-dependent anabaena ribonucleotide reductase and is equally active with the iron-containing ribonucleotide reductase from Escherichia coli. Anabaena thioredoxin-S2 is a good substrate for E. coli thioredoxin reductase. We have compared the structure of Anabaena and E. coli thioredoxins. Clear structural differences between the proteins, compatible with the large evolutionary distance between the organisms, were seen with respect to total amino acid composition, isoelectric point, tryptic peptide maps, and a low immunochemical cross-reactivity. However, both thioredoxins contain a single oxidation-reduction active disulfide bridge with the amino acid sequence: Cys-Gly-Pro-Cys-Lys. The tryptophan fluorescence emission of Anabaena thioredoxin-S2 increases more than 3-fold on reduction to thioredoxin-(SH)2. This behavior is identical with that of E. coli thioredoxin, suggesting a very similar overall folding of homologous molecules.

Ribonucleotide reductase activity in ether-treated cells of Agmenellum quadruplicatum.

Ribonucleotide (cytidine 5'-diphosphate) reductase activity can be detected in situ in cells of the blue-green alga Agmenellum quadruplicatum, strain PR-6, after the cells are made permeable by treatment with ether. The Agmenellum reductase resembles the enzyme from Escherichia coli.

Activity of the natural algicide, cyanobacterin, on angiosperms.

Cyanobacterin is a secondary metabolite produced by the cyanobacterium (blue-green alga) Scytonema hofmanni. The compound had previously been isolated and chemically characterized. It was shown to inhibit the growth of algae at a concentration of approximately 5 micromolar. Cyanobacterin also inhibited the growth of angiosperms, including the aquatic, Lemna, and terrestrial species such as corn and peas. In isolated pea chloroplasts, cyanobacterin inhibited the Hill reaction when p-benzoquinone, K(3)Fe(CN)(6), dichlorophenolindophenol, or silicomolybdate were used as electron acceptors. The concentration needed to inhibit the Hill reaction in photosystem II was generally lower than the concentration of the known photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. Cyanobacterin had no effect on electron transport in photosystem I. The data indicate that cyanobacterin inhibits O(2) evolving photosynthetic electron transport in all plants and that the most probable site of action is in photosystem II.

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119.

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.

Direct electrochemistry of thioredoxins and glutathione at a lipid bilayer-modified electrode.

By using direct electrochemical analysis we have established that the reduction of Escherichia coli thioredoxin (EcT), T4 thioredoxin (T4T), and glutathione (GSSG) occurs at a self-assembled lipid bilayer-modified gold electrode via two separate one-electron processes. The first electron transfer has half-wave potentials of -0.05 +/- 0.01, -0.07 +/- 0.01, and -0.06 +/- 0.01 V, whereas the second one has values of -0.48 +/- 0.01, -0.39 +/- 0.01, and -0.45 +/- 0.01 V, for EcT, T4T, and GSSG, respectively. The scan-rate dependence of the cyclic voltammetry indicates, for both waves, that the process of electron transfer is dominated by a bulk diffusion of free species to and from the electrode, and that strongly adsorbed species do not significantly contribute at the scan rates used. The voltage separation of the peak currents indicates a quasi-reversible electron transfer process with an electrochemical rate constant which is larger for the second (lower potential) electron than for the first one. Using the above half-wave potentials of the one-electron steps, one can calculate a thermodynamic half-wave potential for the two-electron reduction processes. The values of these potentials are -0.265, -0.23, and -0.25 V for EcT, T4T, and GSSG, respectively. These are in excellent agreement with literature values obtained from equilibrium measurements of enzyme-catalyzed reactions involving these species. It is quite clear from these results that lipid bilayer-modified electrodes provide a biocompatible and direct means of efficiently carrying out electrochemical reactions with sulfur-based redox systems, as we have previously shown to be the case with metalloproteins.

Structural and functional relations among thioredoxins of different species.

Three-dimensional models have been constructed of homologous thioredoxins and protein disulfide isomerases based on the high resolution x-ray crystallographic structure of the oxidized form of Escherichia coli thioredoxin. The thioredoxins, from archebacteria to humans, have 27-69% sequence identity to E. coli thioredoxin. The models indicate that all the proteins have similar three-dimensional structures despite the large variation in amino acid sequences. As expected, residues in the active site region of thioredoxins are highly conserved. These include Asp-26, Ala-29, Trp-31, Cys-32, Gly-33, Pro-34, Cys-35, Asp-61, Pro-76, and Gly-92. Similar residues occur in most protein disulfide isomerase sequences. Most of these residues form the surface around the active site that appears to facilitate interactions with other enzymes. Other structurally important residues are also conserved. A proline at position 40 causes a kink in the alpha-2 helix and thus provides the proper position of the active site residues at the amino end of this helix. Pro-76 is important in maintaining the native structure of the molecule. In addition, residues forming the internal contact surfaces between the secondary structural elements are generally unchanged such as Phe-12, Val-25, and Phe-27.

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.

Reduction of mutant phage T4 glutaredoxins by Escherichia coli thioredoxin reductase.

Fifteen mutant T4 glutaredoxins (previously T4 thioredoxin) have been assayed for activity with Escherichia coli thioredoxin reductase. The mutations include substitutions in the region of the active site, in the 2 cysteines, and in the 2 residues between the cysteines forming the active-site disulfide bridge. Mutant thioredoxins where substitutions have been made in charged residues around the active site show the biggest differences in activity. The positive residues Lys-13 and Lys-21 were found to be important for efficient binding to thioredoxin reductase. Substitution of the aspartic acid at position 80 with a serine produced a glutaredoxin with superior activity. This mutant glutaredoxin has earlier been shown to be more efficient than the wild type in thiol transferase activity (Nikkola, M., Gleason, F. K., Saarinen, M., Joelson, T., Björnberg, O., and Eklund, H. (1991) J. Biol. Chem. 266, 16105-16112). Even the glutaredoxin P66A, where the active-site cis-proline has been substituted, could be efficiently reduced by thioredoxin reductase. Glutaredoxins lacking one or both cysteines were not active.