User:Emma Ste.Marie/Sandbox 1: Difference between revisions

Thioredoxin reductases (TrxRs) are a family of pyridine nucleotide-disulphide oxidoreductases that catalyze the reduction of thioredoxin, a class of small redox proteins known to be present in all organisms. Thioredoxin (Trx) plays a role in many important biological processes, including redox signalling. There are two classes of TRs that differ by size and the number of redox centers present in the enzyme. The lower molecular weight (Mr) TrxRs have two redox centers; a flavin adenine dinucleotide (FAD/H) and an N-terminal disulfide redox center[1]. Bacteria, plants, archaea and most unicellular eukaryotes utilize this low Mr, non-selenoprotein form of TrxR. In contrast, the mammalian-type TrxR (mTrxR) carries a C-terminal disulfide redox center in addition to the FAD/H and N-term disulfide redox centers. These higher Mr mTrxRs contain the selenium-containing amino acid selenocysteine (Sec)[1]. There are three human Sec-containing thioredoxin reductases: a cystolic form (type 1, mTrxR1), a mitochondrial form, and a specialized testes specific enzyme[2].

The Trx/mTrxR system is essential for maintaining redox homeostasis; there are numerous systems with thiol-dependent redox mechanisms, which are related to important pathological states and human diseases. Trx/mTrxR system plays a role in the following pathways: apoptosis, DNA replication and repair, protein repair, oxidative stress defence, redox signaling, cell cycle arrest, transcription, protein folding, and glucose metabolism. Disregulation of the Trx/mTrxR system is closely linked to many human diseases including: cardiovascular diseases, diabetes, cancer, aging, inflammation, virus infection, rheumatoid arthritis, and many others.

Three types of mTrxRs have been characterized: the cytosolic form (mTrxR1), the mitochondrial form (mTrxR2), and thioredoxin glutathione reductase (TGR) that is found in testes. TGR is a TrxR-glutathione reductase hybrid enzyme that possesses an N-terminal glutaredoxin-domain in addition to a C-terminal selenocysteine-containing redox centre[5]. Cytosolic mTrxR1s contain a C-terminal redox center with the sequence Xaa-Cys1-Cys2-Xaa (Cys-mTrxRs), or, Xaa-Cys-Sec-Xaa (Sec-mTrxRs)[6]. In contrast, mTrxR2s (the mitochondrial form), contain a C-terminal disulfide redox center with the sequence: Gly-Cys1-Gly-Gly-Gly-Lys-Cys2-Gly[6]. mTrxRs belong to a family of functional homodimeric pyridine nucleotide disulphide oxidoreductases with high homology to glutathione reductases (GRs) and lipoamide dehydrogenase (LipDH). The overall fold of mTrxR1 resembles other homodimeric pyridine nucleotide disulphide oxidoreductases[5]. mTrxRs function as head to tail homodimers, and each subunit contains a binding domain for FAD and NADPH, an interface domain, and a discrete active centre[6]. Currently, there are no crystal structures of the flexible C-terminal redox centre of mTrxR1 without substrate present. However, crystal structures of <scene name='78/785330/Mtrxr1_bound_to_trx/1'>mTrxR1 bound to Trx</scene> have been solved.

The first crystal structure of Type 1 mTrxR in complex with its Trx substrate was solved by Karin Fritz-Wolf, et al. in 2011 and gave much information into structural details of the enzyme. The N-terminally located redox active disulfide (Cys59-Cys64) is buried in the protein, whereas the second, C-terminally located redox active selenosulfide (Cys497-Sec498) is positioned on a flexible, highly accessible C-terminal tail of the other subunit[5]. Complex formation of mTrxR1 with Trx involves the generation of an intermolecular disulphide bond between the catalytic residues Cys32 of Trx and Sec498 of mTrxR1, which is stabilized by several other interactions. Some of the other interactions include: hydrophobic packing (mainly Trp resudies buried with their indole rings at the interface region), hydrogen bonds between amino acid side chain residues, and other interactions involving several oppositely charged residues (electrostatic interactions). All residues involved in forming the intermediate are highly conserved in mammals[5]. It is important to note in this crystal structure that penultimate Sec residues (one on each monomer) were mutated to Cys residues (''E. coli'', the expression vector used, does not have the machinery to incorporate Sec into proteins). Additionally, the resolving Cys residue was mutated to alanine to keep Trx bound.