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 homo- geneous 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)z is a hydrogen donor for the adenosylcobalamin-dependent Anabaena ribonucleotide reductase and is equally active with the iron-con- taining ribonucleotide reductase from Escherichia coli. Anabaena thioredoxin-Sz is a good substrate for E. coli thioredoxin reductase. We have compared the structure of Anabaena and E. coli thioredoxins. Clear struc- tural 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 im-munochemical cross-reactivity. However, both thiore- doxins contain a single oxidation-reduction active disulfide bridge with the amino acid sequence: Cy;-Gly-Pro-Cys-Lys. The tryptophan fluorescence emission of Anabaena thioredoxin-Sz increases more than %fold on reduction to thioredoxin-(SH)z. This behavior is iden- tical with that of E. coli thioredoxin, suggesting a very

and also offer additional data on proteins and evolution comparable to that provided by cytochrome c (3).
Other than the protein from E. coli, no bacterial thioredoxin has been isolated and described as to structure and reactivity. In this paper we report the purification and some of the properties of thioredoxin from the filamentous cyanobacterium Anabaena 7119. The ribonucleotide reductase from this organism has recently been purified and shown to be an adenosylcobalamin-dependent reductase (4) comparable to that found in Lactobacillus leichmannii and other prokaryotes (5). This is in marked contrast to the iron-containing reductase found in E. coli and mammals (6). The data here reported show that the cyanobacterial thioredoxin is homologous to the protein from E. coli despite the large evolutionary distance between these organisms.

DISCUSSION
This paper describes the isolation of a homogeneous thioredoxin from Anabaena sp. and some of its characteristics. Thioredoxins have also been studied from E. coli (l), Lactobacillus leichmannii (26), the green alga Scenedesmus obliquus (27), yeast (24), and mammalian liver (25, 28). All these thioredoxins have certain properties in common. They are heat-stable proteins with molecular weights of approximately 12,000. In the reduced form they serve as hydrogen donors for E, coli as well as their homologous ribonucleotide reductases (6). This indicates identical thiol oxidation-reduction mechanisms and thus the same active center structure. On the other hand, the thioredoxins differ in their reactivity with thioredoxin reductases or antibodies, consistent with marked primary structural differences. A summary of these points is given in Table 111.
Only thioredoxin from E. coli has been extensively characterized. Results from an x-ray crystallographic investigation to 2.8-A resolution demonstrate that this molecule represents a novel type of enzyme structure (29); the active center disulfide is located at the COOH-terminal end of a P-pleated sheet protruding out into the solution. Furthermore, the molecule consists of two prominent folding domains of secondary structure connected by a short hinge region. The folding of Anabaena thioredoxin-S2 must be very similar to that of E. coli thioredoxin-S2, since both share reactivity properties and are substrates for the otherwise highly specific thioredoxin reductase of E. coli (2). The structure of Anabaena thioredoxin may be represented as shown in Fig. 7. The amino acid sequence of the active center pentapeptide cleaved by trypsin and chymotrypsin digestion is identical in E. coli, Anabaena, and also in yeast thioredoxins (30). The identical tryptophan fluorescence spectra of Anabaena and E. coli thioredoxin-SZ and thioredoxin-(SH)z are a strong argument for placing the tryptophans of Anabaena in the same relative position as in E. coli (Trp-28 and Trp-31) (20). The low quantum yield of tryptophan fluorescence in thioredoxin-S2 is attributed to quenching by the disulfide (21). The large increase in fluorescence on reduction is caused by Trp-28, which moves as the result of a localized conformational change (31).
Despite the identities in overall folding and active center residues, including conservation of thioredoxin reductase binding site(s), the peptide maps and immunological data show that Anabaena and E. coli thioredoxins have quite different primary structures. This is consistent with evolution of homologous proteins. From an evolutionary point of view, the cyanobacteria are considered to be direct descendants of an ancient group of microorganisms (32). The occurrence of an adenosylcobalamin-dependent ribonucleotide reductase in the majority of common cyanobacteria further c o n f i i s their ancient lineage (33). The fact that there exists a high degree of homology between the thioredoxins in a primitive photosynthetic bacterium such as Anabaena and the geneologically distant E. coli (34) indicates that natural selection has conserved the thioredoxins throughout evolution, although the ribonucleotide reductases have diverged widely.
The role of thioredoxin in deoxyribonucleotide synthesis is presently unclear since the discovery of the glutaredoxin system in E. coli (35) and the characterization of thioredoxinnegative mutants (36). However, thioredoxin is known to have other functions. Thioredoxin has been shown to act as a cofactor for the 3"phosphoadenosine 5'-phosphosulfate sulfotransferase system of yeast (37) and cyanobacteria (38). As a regulatory factor for photosynthetic enzymes, three thioredoxin fractions have been purified from spinach (39). Two of these, from chloroplasts, function in specific regulation of enzymes of carbon dioxide fiation, fructose bisphosphatase and NADP-malate dehydrogenase. Both enzymes show increased activity after reduction by the chloroplast thioredoxin (39). Their ability to serve as hydrogen donor for E. coli ribonucleotide reductase, however, is minimal (40). Since the cyanobacteria are believed to be the evolutionary prototypes for chloroplasts (41, 42), the function of the Anabaena thioredoxin system described here in regulation of other aspects of cyanobacterial metabolism merits further investigation.   6. CW-cellulose -1 1.7 a The Teactlon mixture contained 100 W WtaLTSIum phosphate buffer, pH 7.0. 1 mU EDTA, 480 ull NADPH and 1.6 mU InSulin. Reactlon mlxture I500 ul) and thloredoxln fracrlon 11-25 "1) were added to two Cuvettes a t 25OC. The reactlon was lnltlatsd by addition of 12 vg calf thymus thloredoxin reductase to one Cuvette and the Oxldotlon of NADPH was monitored at 340 nm In a Zelss PWQ I11 spectrophotometer wlth an automatlc ampliflcarlon unit and a Servogor recorder.