Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp. strain PCC 7120 genomic DNA. One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherchia coli and Anabaena sp. strain PCC 7119. This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins. One gene was identified in a library and was subcloned into a pUC vector and used to transform E. coli strains lacking functional thioredoxin. The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E. coli.Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and fl. Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids. The Anabaena strain 7120 thioredoxin gene was expressed in E. coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate. The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin. It is a small heat-stable redox protein and an efficient protein disulfide reductase. It is not a substrate for E. coli thioredoxin reductase. Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E. coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts. The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin. However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown.Thioredoxin is a small (Mr, approximately 12,000) heatstable redox protein found in cells of a wide variety of both procaryotes and eucaryotes. The active site is a cystine disulfide with the amino acid sequence --Trp-Cys-Gly-ProCys--. The reduced form was originally described in yeast extracts, in which it acts as a reducing agent for sulfate reductase (2). This protein was subsequently shown to be a hydrogen donor for Escherichia coli ribonucleotide reductase (23), a function which it can also serve in other cells. Thioredoxin has been reported as a cofactor, presumably as reducing agent, for methionine sulfoxide reductase in E. coli (10) and adenosine 3'-P 5'-phosphosulfate transferase (39). It can function as a protein disulfide reductase and has been shown to effectively reduce the disulfides in proteins such as insulin (17) and NADP-dependent malate dehydrogenase (35). The latter activity of thioredoxin may enable it to regulate in vivo enzyme activity via redox control.The dithiol form of thioredoxin is involved in bacteriophage reproduction in E. coli. The T7 gene 5 protein (DNA polymerase) forms a complex with the host thioredoxin (32), and this complex increases the processivity of the polymerase (37). E. coli mutants lacking thioredoxin will not support T...

Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin

“…Thioredoxin from E. coli was prepared by the published procedure [ 161. Mutant thioredoxins D26A [ 171, K36E, R insert [18] and the protein of Anabaena 7120 [19] were obtained from Drs F. K. Gleason and J. A. Fuchs, Uni-versity of Minnesota, St. Paul, USA; E. coli glutaredoxin and thioredoxin P34H [20] were kindly provided by Dr A. Holmgren, Karolinska Institutet, Stockholm, Sweden; spinach chloroplast thioredoxins [9] were gifts of Dr P. Schiirmann, University of Neuchatel, Switzerland.…”

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

“…coli thioredoxin and Anabaena sp. strain PCC 7119 and PCC 7120 thioredoxins (T-1 and T-2, respectively) were purified from E. coli strains containing plasmid-encoded thioredoxins as previously described (1,20). Thioredoxinglutaredoxin produced by bacteriophage T4 and a mutant protein (V15G;Y16P) with the active site sequence altered to that of E. coli thioredoxin (26) were a generous gift of Matti Nikkola, Swedish Agricultural University, Uppsala.…”

“…We had previously reported the occurrence of two thioredoxin genes in the cyanobacterium Anabaena sp. strain PCC 7120 (1). Only one of these was characterized further.…”

Biochemical characterization of thioredoxin 1 from Dictyostelium discoideum

“…The weak interaction of DdTRXl with E. coli NTR reflects the specificity of the latter. While some TRX are readily reduced by NTR (Table l), others are not accepted as substrates, e. g. TRX h from spinach (Florencio et al, 1988), TRX from Anabaena 7120 (Alam et al, 1989) and CHI from C. reinhardtii .…”