Abstract
Indoleamine 2,3-dioxygenase is an immunomodulatory enzyme that shows great promise when delivered exogenously as a protein therapeutic. However, IDO activity is under complex redox control, mediated in part by multiple cysteine residues within its primary sequence. We have characterized three IDO mutants in which solvent-accessible cysteine residues were mutated to chemically-similar serine residues, "IDO(C4S4)" with C112S, C159S, C206S, and C308S mutations and "IDO(C5S3)" with C112S, C159S, and C308S mutations based on prior reports that C206 is necessary for catalytic function, and IDO(C0S8), in which all cysteine residues were mutated to serines. IDO(C0S8) was expressed in poor yield and demonstrated less than 1% activity when compared to wild-type IDO. In contrast, IDO(C4S4) and IDO(C5S3) demonstrated robust enzymatic activity, though IDO(C5S3) had a slower V (max) than wild-type and IDO(C4S4). Computational predictions and experimental measurements suggested a high degree of structural similarity between the wild-type IDO and IDO(C4S4), with subtle perturbation of α-helical content for IDO(C5S3). The structure of IDO(C0S8) was predicted to be significantly different than that of wild-type IDO. IDO(C4S4) and IDO(C5S3) were more stable than wild-type IDO over time at physiological, ambient, and reduced temperatures, likely due to diminished oxidation of the mutant IDO forms. Based on the increased V (max) and robust thermal stability of IDO(C4S4), we fused it to the anchoring moiety galectin 3, to evaluate its effectiveness in a mouse model of psoriasis. The IDO(C4S4)-galectin-3 fusion blunted the rate and severity of disease as compared to wild-type IDO-galectin-3 fusion. When compared to historical data with Cys-Ala IDO mutants, this study highlights the importance of employing amino acid substitution according to similarity in isosteric and isostructural shape to advance IDO as an immunomodulatory therapeutic.