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.

Properties of the endogenous components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125

“…On the other hand, a higher K M (12.3 lM) was reported for the hyperthermophilic TrxR from Aeropyrum pernix (Ap) towards ApTrx (Jeon and Ishikawa 2002). Finally, the reversibility of the electron transfer in the reconstituted thioredoxin system allowed the determination of the E 00 (PhTrx) , whose value (-289 mV) was similar to that recently reported for the recombinant system of P. haloplanktis (E 00 (rPhTrx) = -276 mV; Cotugno et al 2009). Furthermore, no great differences were found with the corresponding parameters related to the mesophile E. coli (E 00 (EcTrx) = -270 mV; Krause et al 1991) or the hyperthermophile A. pernix (E 00 (ApTrx) = -262 mV; Jeon and Ishikawa 2002).…”

“…The activity of PhTrxR was first measured with the synthetic substrate DTNB, using a reaction temperature of 20°C, corresponding to the maximum growth temperature tolerated by P. haloplanktis. In particular, the kinetic parameters of the reduction reaction (k cat , 3 s -1 ; K M for DTNB, 2.6 mM), although not coincident with those previously reported for the recombinant PhTrxR (k cat , 6.9 s -1 ; K M for DTNB, 1.9 mM; Cotugno et al 2009), indicate that the enzyme was purified in its active form. The difference could be explained with the understoichiometric FAD/PhTrxR ratio found in the endogenous flavoenzyme.…”

“…In particular, the components of the thioredoxin system have been characterised in some (hyper)thermophilic archaea, such as Sulfolobus solfataricus (Ruocco et al 2004;Grimaldi et al 2008), Aeropyrum pernix (Jeon and Ishikawa 2002), Pyrococcus horikoshii (Kashima and Ishikawa 2003) and Thermoplasma acidophilum (Hernandez et al 2008). The information available for the thioredoxin system in coldadapted microorganisms is limited to a recent paper, describing the properties of recombinant forms of Trx and TrxR from the psychrotolerant eubacterium Pseudoalteromonas haloplanktis TAC 125 (rPhTrx and rPhTrxR, respectively; Cotugno et al 2009). It is known that this microorganism, isolated from the Antarctic sea and able to grow in a broad range of temperatures (4-20°C), adapts its biochemical machinery to functioning either in the cold or in moderate temperature conditions (Birolo et al 2000;Medigue et al 2005).…”

“…In recent years, the mechanisms leading to the formation/breakdown of disulphide bridges in proteins have been examined also in extremophilic organisms (Jeon and Ishikawa 2002;Kashima and Ishikawa 2003;Ruocco et al 2004;Ladenstein and Ren 2006;Grimaldi et al 2008;Hernandez et al 2008;Cotugno et al 2009). In particular, the components of the thioredoxin system have been characterised in some (hyper)thermophilic archaea, such as Sulfolobus solfataricus (Ruocco et al 2004;Grimaldi et al 2008), Aeropyrum pernix (Jeon and Ishikawa 2002), Pyrococcus horikoshii (Kashima and Ishikawa 2003) and Thermoplasma acidophilum (Hernandez et al 2008).…”

Cloning, Expression, Purification, and Characterization of Glutaredoxin from Antarctic Sea-Ice BacteriumPseudoalteromonassp. AN178

“…Antarctic fungi Penicillium sp. could increase activities of antioxidant enzymes such as catalase and superoxide dismutase for adaptation to the high oxygen concentration [13]. Thioredoxin and thioredoxin reductase from the Antarctic psychrophilic eubacterium Pseudoalteromonas haloplanktis were investigated through the heterologous expression of their genes and the biochemical investigation on the recombinant proteins [14].…”