The thioredoxin system in the psychrophilic eubacteriumPseudoalteromonas haloplanktis

The thioredoxin system, involved in preservation of the reduced state of cytoplasmic proteins, includes two key components of the intracellular redox balance, namely thioredoxin (Trx) and thioredoxin reductase (TrxR). Trx is a small monomeric protein containing the conserved CXXC sequence; a similar motif is also present in the homodimeric TrxR, a NADPH-dependent flavoenzyme. Reduction of protein targets by the Trx/TrxR system involves oscillation between oxidized and reduced form of both cysteine motifs engaged in a reversible disulfide bridge. A previous characterization of Fe-SOD from the psychrophilic eubacterium Pseudoalteromonas haloplkanktis (Ph) proved the high efficient catalysis of this enzyme, another key element of the intracellular redox balance (1). Here we present the properties of the thioredoxin system in P. haloplanktis. PhTrx and PhTrxR were obtained as recombinant proteins and their properties were compared with those of the corresponding endogenous proteins purified from P. haloplanktis. To this aim, genes encoding PhTrx and PhTrxR were cloned in a pET plasmid and as His-tagged recombinant proteins. Exogenous FAD was added in the bacterial total extract to reach a stoichiometric FAD content in the recombinant PhTrxR; under these conditions its activity was almost identical to that measured on endogenous PhTrxR. Kinetic parameters of the dithiobisnitrobenzoate (DTNB) reduction by recombinant and endogenous PhTrxR were determined. Using this assay, thermophilicity and thermostability of the flavoenzyme were also evaluated. Maximum activity was reached at 30°C; however the enzyme showed a marked heat tolerance, its half life being 10 min at 60°C. This latter finding was confirmed by heat denaturation profiles obtained by fluorimetric measurements. The functionality of PhTrx was evaluated by its thiol-disulfide oxidoreductase activity in the presence of DTT as electron donor, and human insulin as substrate. Finally, the combined activity of PhTrxR and PhTrx in a complete thioredoxin system, was also evaluated using NADPH as electron donor and human insulin as the thiored