Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. Melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2.
As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects (Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Müller, H. M., Botella-Munoz, J., Schneuwly, S., Schirmer, R. H., and Becker, K. (2001) Science 291, 643-646). In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 (K(m) = 5.2 microm, k(cat) = 14.5 s(-1)) and in its dithiol form, an electron donor for TPx-1 (K(m) = 9 microm, k(cat) = 5.4 s(-1)). DmTrx-2 is capable of reducing glutathione disulfide with a second order rate constant of 170 m(-1) s(-1) at pH 7.4 and 25 degrees C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass = 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step we have cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae.
Bauer
Holger
H
Biochemistry Center, Im Neuenheimer Feld 328, Heidelberg University, D-69120 Heidelberg, Germany. holger.bauer.pf@gmx.de
Kanzok
Stefan M
SM
Schirmer
R Heiner
RH
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000201:enzymology*; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins; D016415:Sequence Alignment; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C491201:DHD protein, Drosophila; D029721:Drosophila Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C516042:Trx-2 protein, Drosophila; D013879:Thioredoxins; D010544:Peroxidases; D054464:Peroxiredoxins