Protein disulfide oxidoreductase from active sites

Proteins from hyperthermophilic archaea usually contain a decreased number of cysteine residues compared to their mesophilic counterparts. This is probably due to the easy oxidation and instability of sulfur-containing amino acid residues at high temperatures. Consequently, there have been arguments, although with little supportive evidence, that archaeal proteins contain hardly any disulfide bonds. However, a few protein disulfide oxidoreductases have been purified and characterized in archaea. Their existence suggests that thiol-disulfide exchange reactions actually take place in archaea as in bacteria and eukarya. The determination of the first three-dimensional structure of a protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus (P/PDO) has provided the first view of active site disulfides in an archaeal protein. It reveals a unique structural form compared to those of protein disulfide oxidoreductases in bacteria and eukarya. 

Unlike thioredoxin, glutaredoxin, and DsbA, which possess only one thiore-doxin-like motif, P/PDO is the first protein disulfide oxidoreductase whose three-dimensional structure has been shown to contain two thioredoxin fold motifs with two active sites. Thus, P/PDO shows structural resemblance to PDI and PDI-like protein s. This structural feature suggests that P/PDO is probably not just a simple protein disulfide reductant like thioredoxin as described previously. It may belong to the growing family of PDI-like proteins. From a structural point of view, P/PDO may represent the simplest form of PDI. 

TrxRs are homodimeric flavoproteins [80] that catalyze the NADPH-dependent reduction of thioredoxin (Trx), a ubiquitous 12 kDa protein that is the major protein disulfide reductase in cells [81], and belongs to the pyridine nucleotide-disulfide oxidoreductase family [82]. Each monomer includes an FAD prosthetic group, a NADPH binding site and an active site containing a redox-active selenol group. Electrons are transferred from NADPH via FAD to the active-site selenol of TrxR, which then reduces the substrate Trx [83]. The crystal structure of TrxR is shown in Fig. 13 [84],... 

Thioredoxin behaves differently from other proteins. Because of the great interest of the sulphur function in this enzyme (it is the active site of this thiol-disulfide oxidoreductase), the reduction of oxidized thioredoxin was studied in detail (139, 140). The disulfide radical is much more acidic than in other proteins, and its decay leads only to the reduced protein. Site-directed mutagenesis was used to modify selectively two amino-acids, Asp30 and Trp35, in order to observe the modulation of the redox properties of the sulphur functions. It was thus shown that both residues play a role in the proton transfer associated to electron transfer, although differently for instance, removal of W35 increases the pKa of... 

Unfortunately, such methods require the exact placement of atoms within protein side chains and are inapplicable to the inexact, low-resolution predicted structures generated by the state-of-the-art ab initio folding and threading algorithms (see Sections IV-VI). These methods are required when the sequence identity of the sequence of interest to solved structures is too low to use comparative modeling. To address this need, Skolnick and Fetrow have recently developed fuzzy, inexact descriptors of protein functional sites [8]. They are applicable to both high-resolution, experimental structures and low-resolution (backbone RMSD 4—6 A from native) structures. These descriptors are a-carbon-based, fuzzy functional forms (FFFs). Initially, they created FFFs for the disulfide oxidoreductase [8,10] and a/p-hydrolase catalytic active sites [11] (an additional 198 have now been bmlt, with comparable results [234]). 

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