title: Differential cold-adaptation among protein components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125.
abstract: Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.
authors.affiliation: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131, Napoli, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Cotugno
authors.forename: Maria
authors.initials: M
authors.lastname: Rosaria Ruocco
authors.forename: Salvatore
authors.initials: S
authors.lastname: Marco
authors.forename: Patrizia
authors.initials: P
authors.lastname: Falasca
authors.forename: Giovanna
authors.initials: G
authors.lastname: Evangelista
authors.forename: Gennaro
authors.initials: G
authors.lastname: Raimo
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.forename: Mariorosario
authors.initials: M
authors.lastname: Masullo
authors.forename: Emmanuele
authors.initials: E
authors.lastname: De Vendittis
mesh_terms: D000222:Adaptation, Physiological / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D005051:Eubacterium / Q000378:metabolism*; D031161:Pseudoalteromonas / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase