title: Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii.
abstract: A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.
authors.affiliation: Department of Biochemistry, University of Minnesota, Minneapolis.
authors.forename: S C
authors.initials: SC
authors.lastname: McFarlan
authors.forename: H P
authors.initials: HP
authors.lastname: Hogenkamp
authors.forename: E D
authors.initials: ED
authors.lastname: Eccleston
authors.forename: J B
authors.initials: JB
authors.lastname: Howard
authors.forename: J A
authors.initials: JA
authors.lastname: Fuchs
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000032:analysis* / Q000201:enzymology / Q000254:growth & development; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013880:Thioredoxin-Disulfide Reductase / Q000302:isolation & purification; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification*; D014357:Trypsin
chemical_list: D001426:Bacterial Proteins; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D002918:Chymotrypsin; D014357:Trypsin