Studies on human indoleamine 2,3-dioxygenase (IDO)

Author

Tamantha Kim Littlejohn, University of Wollongong

Year

2001

Degree Name

Doctor of Philosophy

Department

Faculty of Science

Recommended Citation

Littlejohn, Tamantha Kim, Studies on human indoleamine 2,3-dioxygenase (IDO), Doctor of Philosophy thesis, Faculty of Science, University of Wollongong, 2001. https://ro.uow.edu.au/theses/1872

Abstract

L-Tryptophan is an essential amino acid and the least abundant amino acid in humans. An understanding of the metabolism of L-tryptophan is therefore physiologically important. The enzyme indoleamine 2,3-dioxgyenase (IDO) catalyses the first, and rate limiting, step in the major pathway of tryptophan metabolism, the kynurenine pathway. The physiological role of IDO is not fully understood, but is of great interest because IDO is a widely distributed enzyme in humans and can alter the levels of tryptophan, which is vital for cell growth. Induction of IDO may therefore have a protective role in bacterial, viral and protozoan infections. Additionally, tryptophan metabolism via the kynurenine pathway produces a number of compounds that are neurotoxic and have been implicated in neurodegenerative disease. Products of this pathway have also been shown to be involved in lens aging and possibly age-related cataract formation. Although implicated in a number of disease processes, the exact function of IDO is poorly understood. The elucidation of the structure of this important human enzyme will assist in determining its function in the body. Structural analysis will aid the design of specific inhibitors that may contribute to the understanding of the pathophysiology or provide treatment leads for the diseases in which the kynurenine pathway has been implicated.

Human IDO can be isolated from tissues such as the placenta, but only in small quantities. Thus a reliable and efficient source of functional IDO was required to provide sufficient protein for structural analysis. This study details the development of a recombinant expression system for human IDO. The expression construct, Escherichia coli EC538 (pREP4, pQE9-IDO) produced a full-length human IDO protein, with a N-terminal hexahistidyl tag. The molecular weight of the recombinant protein product was determined to be 46, 976 Da and it has a specific activity of 149 Hmole kynurenine produced per hour per mg of enzyme, with L-tryptophan as the substrate. The recombinant protein contained 0.8 moles of heme per mole of enzyme and the isoelectric point was determined to be 7.1. As with the native enzyme, recombinant IDO exhibited characteristic maxima at 406 and 630 nm by ultravioletvisible spectroscopy and these were shifted to 420 and 560 nm in the reduced form. The recombinant enzyme used L-tryptophan, D-tryptophan and 5-hydroxy-L-tryptophan as substrates. The K_m values were determined to be 20 ,μM for L-tryptophan, 5 mM for D-tryptophan and 440 μM for 5-hydroxy-L-tryptophan. The maximum turnover rates (V_max) for these substrates were 120 (L-tryptophan), 160 (D-tryptophan) and 5.3 (5- hydroxy-L-tryptophan) moles of kynurenine produced per hour per mole of enzyme. These were similar to the native enzyme, indicating that the addition of a hexahistidyl tag did not significantly affect the catalytic activity of the enzyme. It could be concluded from the characterisation of this purified recombinant protein that it was essentially identical to the native human enzyme, in terms of spectroscopic properties, cofactor dependency, substrate specificity and molecular activity. Circular dichroism spectroscopy showed that recombinant human IDO consists of a large random coil component, with approximately 30% alpha helical and 10% beta sheet structure. Initial X-ray crystallographic studies of the recombinant protein resulted in the formation of small crystals, which is promising in the quest to solve the 3-dimensional structure of human IDO.

To identify amino acid residues important in substrate or heme binding in IDO, site-directed mutagenesis was undertaken. It had been proposed that a histidine acts as the proximal heme ligand in IDO. Mutation of the only three highly conserved histidines in EDO species, His¹⁶ , His³⁰³ and His³⁴⁶ , was therefore conducted. Of these, only His³⁴⁶ was shown to be essential for heme binding, indicating that this histidine residue may be the proximal ligand, and suggesting that neither His³⁰³ nor His¹⁶ acts as the proximal ligand. Site-directed mutagenesis of Asp²⁷⁴ compromised the ability of IDO to bind heme. This indicates that Asp²⁷⁴ may also coordinate to the heme and that it could be the distal ligand or be essential in maintaining the conformation of the heme pocket. This is the first study to show that both His³⁴⁶ and Asp²⁷⁴ are essential for the binding of heme in human IDO.

Site-directed mutagenesis also facilitated the production of a truncated IDO protein. This truncated IDO maintained enzymatic activity, indicating that the C-terminal region of IDO is not required for activity and may be a flexible region. The removal of this region in the truncated recombinant human IDO may be beneficial for future X-ray crystallographic studies.

An expression and purification system has therefore been established that produces an enzymatically active recombinant EDO, which is comparable to the native enzyme. Access to this enzyme will greatly aid the design of EDO inhibitors, which may assist in the elucidation of the role(s) of IDO in humans. Site-directed mutagenesis has identified two residues, His³⁰³ and Asp²⁷⁴ that are essential for heme binding, and thus enzymatic activity, in human IDO.