Recombinant human indoleamine 2,3-dioxygenase

Typescript."December 2009"A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Macquarie University, 2009.Thesis (PhD)--Macquarie University, Faculty of Science, Dept. of Chemistry and Biomolecular Sciences, 2009.Bibliography: leaves 169-205.1. Introduction -- 2. Expression and purification of indoleamine 2,3-dioxygenase -- 3. Characterisation of indoleamine 2,3-dioxygenase -- 4. Re-incorporation of heme into indoleamine 2,3-dioxygenase -- 5. Mutagenesis of indoleamine 2,3-dioxygenase -- 6. Conclusions and recommendations -- 7. Experimental -- AppendicesIndoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase that catalyses the first and rate-limiting step in the kynurenine (Kyn) pathway of L-tryptophan (L-Trp)catabolism. Attention has been focused on IDO because the Kyn metabolic pathway is involved in a variety of physiological functions and diseases. In this study the expression and purification of recombinant human indoleamine 2,3-dioxygenase (rhIDO) in E. coli(pQE-9-IDO, pREP4) was investigated in order to obtain high quality enzyme in high yields. This study concluded that optimisation could still be achieved by lowering the growth temperature from 37 °C to 30 °C and reduction of IPTG induction from 100 μMto 10 μM. Better yields of rhIDO were obtained when the expression was carried out under dark conditions without the addition of PMSF, addition of hemin prior to lysis of the cells, increasing the NaCl concentration from 150 mM to 500 mM and lowering the imidazole variation from 10, 30, 40, 65, 80, 90, 190 to 10, 30, 60, 300 mM in the purification through Ni-NTA.Evaluation of the characteristics and stability of rhIDO in order to understand its behaviour as a support towards further studies showed that rhIDO activity and yield under certain conditions of purification were significantly decreased. Further studies concluded that the loss of rhIDO activity was most probably due to the loss of the heme prosthetic group of the enzyme or the non effectiveness of the heme-protein complex. A study with hemin to examine the possibility of re-incorporation of heme showed moderate re-incorporation. It was concluded, however, that keeping the heme intact through the supplementation of hemin prior to lysis is a much better alternative for preserving the activity of the enzyme. Comparison studies between rhIDO and rmIDO showed that the relative catalytic efficiency of rhIDO towards L-Trp (3.73) was higher compared to rmIDO (1.40) whereas the relative catalytic efficiency of rhIDO towards D-Trp (0.02) was lower compared to rmIDO (0.03). CD and thermal melt studies established that rhIDO is less helical (61% helix) than rmIDO (71% helix). The studies also showed that rhIDO has a lower melt temperature (50 ºC) than rmIDO (65 ºC)indicating that rmIDO is more thermostable than rhIDO.The contribution of individual cysteines towards the overall catalytic properties and stability of the rhIDO was evaluated through mutagenesis studies. This was done by comparing the resulting outcomes from the C126A, C286A, C322A and C349A rhIDO mutations towards that of the non-mutated rhIDO. All the mutants exhibited a decrease in specificity towards L-Trp with C322A showing the highest decrease of 78% loss. However, with D-Trp, mutants C126A and C322A showed increased specificity whereC126A showed the highest increase (280%). Mutants C286A and C349A showed decreased activity with C349A having the most (67% loss). This study confirms that the Cys to Ala site-directed mutagenesis contributes to the changes in the kinetics of the mutated rhIDO, and that the cysteine moieties of rhIDO are involved in the normal catalytic function of the enzyme. It is highly likely that the change from Cys to Ala in the mutants changed the conformation of the enzyme, which was a determining factor to the accessibility of the specific substrates to the active site. This change in conformation resulted in diverse kinetics observed.Mode of access: World wide web1 online resource (xxiv, 222 leaves, bound) ill. (some col.), chart

Mutation of cysteine residues alters the heme-binding pocket of indoleamine 2,3-dioxygenase-1

The hemoprotein indoleamine 2,3-dioxygenase-1 (IDO1) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. Interest in IDO1 continues to grow, due to the ever expanding influence IDO1 plays in the immune response. This study examined the contribution of all individual cysteine residues towards the overall catalytic properties and stability of recombinant human IDO1 via mutagenesis studies using a range of biochemical and spectroscopic techniques, including in vitro kinetic assessment, secondary structure identification via circular dichroism spectroscopy and thermal stability assessment. Upon mutation of cysteine residues we observed changes in secondary structure (principally, shifting from α-helix/β-sheet features to random coil structures) that produced out of plane heme torsion and puckering, changes to thermal stability (including gains in stability for one mutant protein) and differences in enzymatic activity (such as, increased ability to convert non-natural substrates, e.g.d-tryptophan) from wild type IDO1 enzyme

Natural product-inspired pyranonaphthoquinone inhibitors of indoleamine 2,3-dioxygenase-1 (IDO-1)

A series of pyranonaphthoquinone derivatives possessing structural features present in both natural products annulin B and exiguamine A have been shown to exhibit low micromolar inhibition of indoleamine 2,3-dioxygenase-1 (IDO-1). These inhibitors retain activity against the enzyme in a cellular context with an approximate one-log loss of dose potency against IDO-1 in cells. One particular analogue, triazole 8 shows good inhibition of IDO-1 along with little loss of cell viability at low drug concentrations. These results have extended the naphthoquinone series of novel IDO-1 inhibitors based on lead compounds from nature.10 page(s

Mouse and human indoleamine 2,3-dioxygenase display some distinct biochemical and structural properties

The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the most significant pathway for mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its dual role in immunity and the pathogenesis of many diseases. Reported here are differences and similarities between biochemical behaviour and structural features of recombinant human IDO and recombinant mouse IDO. Significant differences were observed in the conversion of substrates and pH stability. Differences in inhibitor potency and thermal stability were also noted. Secondary structural features were broadly similar but variation between species was apparent, particularly in the α-helix portion of the enzymes. With mouse models substituting for human diseases, the differences between mouse and human IDO must be recognised before applying experimental findings from one system to the next.8 page(s

Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase

The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its role in the pathogenesis of many diseases. Here, we report attempts to improve soluble expression and purification of hexahistidyl-tagged recombinant human IDO from Escherichia coli (EC538, pREP4, and pQE9-IDO). Significant formation of inclusion bodies was noted at the growth temperature of 37 °C, with reduced formation at 30 °C. The addition of the natural biosynthetic precursor of protoporphrin IX, δ-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 °C and purification by nickel–agarose and size exclusion chromatography, resulted in protein with 1 mol of heme/mol of protein and a specific activity of 160 μmol of kynurenine/h/mg of protein (both identical to native human IDO). The protein was homogeneous in terms of electrophoretic analysis. Yields of soluble protein (3–5 mg/L of bacterial culture) and heme content are greater than previously reported