The Oxidation of L-Tryptophan in Biology by Human Heme Dioxygenases
thesisposted on 2010-07-26, 14:44 authored by Bibi Sara Ameena Rafice
Tryptophan is an essential amino acid, which is catabolised via the kynurenine pathway leading to the formation of NAD. The initial and rate-limiting step of the kynurenine pathway is controlled by two hemoproteins called tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which oxidise tryptophan to form N-formyl-kynurenine. A bacterial expression system for hTDO was characterised in Chapter 2. Kinetic, spectroscopic and redox analyses determined that significant differences exist. It was found that hTDO does not form a stable ferrous-oxy complex and that the ferric form of the enzyme was catalytically active. In addition hTDO does not discriminate against substrate binding to the ferric derivative. Site-directed mutagenesis of several active site residues and the role of each residue on substrate binding and in catalysis was examined in Chapter 3. The H76 residue was found to be involved in substrate binding. The phenylalanine variants of TDO showed that the hydrophobicity in the active site was essential for catalytic activity. Furthermore, the data showed that F72 is an important substrate binding residue. The highly conserved arginine residue was deemed to be an essential catalytic residue involved in substrate binding. The implications of these findings are discussed in terms of the current understanding of dioxygenase catalysis. In Chapter 4, it was shown that 1-methyl-tryptophan was a substrate for wild type IDO and variants of TDO and IDO, which is contrary to previous findings in dioxygenase literature. No activity was observed for wild type hTDO. Previous crystallographic studies and modelling in combination with kinetic, spectroscopic and redox analyses indicated that the distal histidine in the TDO active site causes steric clashes with 1-methyl-L-tryptophan. This information indicated exclusion of the deprotonation of the indole N1 mechanism, and an alternative reaction mechanism was presented. A new expression system for IDO with a cleavable hexahistidyl tag was constructed in Chapter 5. The analyses indicated that the recombinant protein had the same characteristics as all other mammalian IDOs and that diffraction quality crystals can be grown from the system. Crystal structures of IDO are required to further the understanding of substrate binding and the catalytic mechanism.