Characterization of Escherichia coli-Anabaena sp. hybrid thioredoxins.
Thioredoxin is a small redox protein with an active-site disulfide/dithiol. The protein from Escherichia coli has been well characterized. The genes encoding thioredoxin in E. coli and in the filamentous cyanobacterium Anabaena PCC 7119 have been cloned and sequenced. Anabaena thioredoxin exhibits 50% amino acid identity with the E. coli protein and interacts with E. coli enzymes. The genes encoding Anabaena and E. coli thioredoxin were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate hybrid genes, coding for two chimeric thioredoxins. These proteins are designated Anabaena-E. coli (A-E) thioredoxin for the construct with the Anabaena sequence from the N-terminus to the middle of the active site and the E. coli sequence to the C-terminus, and E. coli-Anabaena (E-A) for the opposite construct. The gene encoding the A-E thioredoxin complements all phenotypes of an E. coli thioredoxin-deficient strain, whereas the gene encoding E-A thioredoxin is only partially effective. Purified E-A thioredoxin exhibits a much lower catalytic efficiency with E. coli thioredoxin reductase and ribonucleotide reductase than either E. coli or Anabaena thioredoxin. In contrast, the A-E thioredoxin has a higher catalytic efficiency in these reactions than either parental protein. Reaction with antibodies to E. coli and Anabaena thioredoxins shows that the antigenic determinants for thioredoxin are located in the C-terminal part of the molecule and retain the native conformation in the hybrid proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012264:Ribonucleotide Reductases / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D013879:Thioredoxins; D012264:Ribonucleotide Reductases