|Institution:||University of Oslo|
|Full text PDF:||https://www.duo.uio.no/handle/10852/11433
Thioredoxin (Trx) is a small ubiquitous protein containing two conserved cysteines that can reversibely form a redox-active disulfide bond, belonging to the Trx superfamily. Ribonucleotide reductase (RNR) was the first enzyme discovered to use Trxs and glutaredoxins (Grxs) for the reduction of active site cysteines. Class Ib RNR uses in many cases a protein called NrdH-redoxin for this purpose. Thiol-disulfide oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate protein. Although most Trx-like proteins do not have a high level of sequence similarity, all enzymes share an overall // sandwich fold, in addition to the conserved C-X-X-C motif. The fundamental reaction mechanism for electron transfer from Trx to its substrate was proposed by Kallis and Holmgren in 1980. As a result of the lowered pKa value observed for the N-terminal cysteine thiol (-SH) in the Escherichia coli Trx C-G-P-C motif, it was suggested that this thiolate (-S-) could perform the initial nucleophilic attack on the substrate disulphide bond. In order for the second nucleophilic attack performed by the buried C-terminal cysteine to take place, deprotonation caused by a conserved aspartate residue in the vicinity of the active site has been proposed. Examples of groups of proteins not encompassing this Asp26, but possessing Trx functionality, are the E. coli NrdH-redoxins, described as the reductants of NrdE of bacterial class Ib RNR, and C. pasteurianum Cp9-redoxins, involved in the reduction of various hydroperoxide substrates. A protein homologous to the NrdH-redoxins and Cp9-redoxins has been located in the Bacillus cereus genome, showing significant amino acid sequence similarity with both of the above mentioned redoxins. However, its function is still unknown. A conserved threonine residue, Thr8, adjacent to the active site is believed to influence the protonation state of the C-terminal cysteine in the active site of this small Trx. Other residues might influence the protonation state of this residue as well. The function and structure of this enzyme, BC3987, has been characterized using various biochemical techniques. The crystal structures of two mutant proteins; BC3987 D11W and T53A, in addition to the native protein, have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values and redox potentials of active site cysteine thiols/disulfides were performed.