The Purification, Cloning, and High Level Expression of a Glutaredoxin-like Protein from the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

A protein has been purified to homogeneity from crude extracts of the hyperthermophilic archaeon Pyrococcus furiosus based on its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol; the protein has a molecular mass of 24.8 kDa and a pI of 4.9, and it is highly heat-stable. The first 29 amino acid residues at the N terminus of the P. furiosus protein were determined by Edman degradation, and its gene was cloned in Escherichia coli. The amino acid sequence derived from the DNA sequence contains the CPYC sequence, which is typical of the active site of glutaredoxin (also called thioltransferase). The C-terminal portion of the P. furiosus protein, containing the conserved sequence, shows sequence similarity with glutaredoxins from different sources. The P. furiosus protein can reduce disulfide bonds in L-cystine in the presence of GSH (the thioltransferase activity) with an optimum pH of 8.0. The expression of the P. furiosus protein, with full activity, in E. coli at a very high level (21% of total soluble protein) is described; the recombinant protein was purified to homogeneity by merely two successive heat treatments and gel filtration chromatography. The features of the P. furiosus protein here described are discussed in light of the current knowledge about the ubiquitous family of protein disulfide oxidoreductases.

Posttranslational Protein Modification inArchaea

“…In addition, a homologous protein had been purified earlier from Sulfolobus solfataricus (150) and was predicted to exist in other species, based upon examination of the genome sequences of hyperthermophilic Archaea (339). However, the homologous protein from Pyrococcus horikoshii together with a second protein identified as a thioredoxin reductase were shown to function as a thioredoxin system, mediating electron transfer from a thioredoxin reductase-like flavoprotein to a protein disulfide bond, suggesting a role for this protein other than as a disulfide bond-introducing PDI (205).…”

Characterization of a Thioredoxin-Thioredoxin Reductase System from the Hyperthermophilic BacteriumThermotoga maritima

“…T. maritima Trx has two redox-active motifs, CPYC and CQYC, which resulted in its annotation as a Grx-like protein (33). In contrast to the lack of homology to conventional Trxs, the primary structure of T. maritima Trx has high levels of identities to some protein disulfide oxidoreductases (PDO) that have molecular masses of around 25 kDa or higher and two redox-active motifs (CXXC and CXXC), which are exclusively isolated from hyperthermophiles, such as P. horikoshii (22), P. furiosus (14), Aquifex aeolicus (35), S. solfataricus (15), and A. pernix (21). T. maritima Trx exhibited both Trx and Grx (thioltransferase) activities, similar to the archaeal PDO (36).…”

Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus : Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase

“…Attempts to stabilize P. furiosus MBX are in progress. In addition to NSR and MBX, the DNA microarray data (Table 1) show that the expression of the gene encoding a glutaredoxin-like protein termed protein disulfide oxidoreductase (PDO) (12,25), PF0094, is up-regulated almost fourfold as part of the primary response to S 0 (Table 1). A specific role for PF0094 as a PDO in P. furiosus has yet to be established.…”

“…Since homologs of PDO and thioredoxin reductase (and RNR) are widespread throughout the archaea, including those that do not utilize S 0 (25,35), it would seem unlikely that PDO has a specific role in reducing S 0 . To investigate this, the recombinant form of the PF0094 protein was obtained using published procedures (12). However, the purified protein had no effect on the S 0 reduction activity of purified NSR activity or on the ability of P. furiosus membranes to couple ferredoxin oxidation to the reduction of NADP or S 0 (data not shown).…”