title: In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.
abstract: Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).
authors.affiliation: Biochemistry and Biophysics Group, Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119-3105, USA. joshuap@amgen.com
authors.forename: Josh T
authors.initials: JT
authors.lastname: Pearson
authors.forename: Sophia
authors.initials: S
authors.lastname: Siu
authors.forename: David P
authors.initials: DP
authors.lastname: Meininger
authors.forename: Larry C
authors.initials: LC
authors.lastname: Wienkers
authors.forename: Dan A
authors.initials: DA
authors.lastname: Rock
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D045303:Cytochromes b / Q000494:pharmacology*; D004789:Enzyme Activation; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008751:Methylene Blue / Q000494:pharmacology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014364:Tryptophan / Q000494:pharmacology
chemical_list: D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007211:Indoles; D029701:Saccharomyces cerevisiae Proteins; C030374:indole; D014364:Tryptophan; D045303:Cytochromes b; D009251:NADPH-Ferrihemoprotein Reductase; D008751:Methylene Blue