Dataset Information

UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1.

ABSTRACT: Human indoleamine 2,3-dioxygenase 1 (IDO) is a heme enzyme that catalyzes the first reaction of the main metabolic pathway of L-tryptophan (Trp) to produce N-formylkynurenin. The reaction involves cleavage of the C2=C3 bond in the Trp indole ring and insertion of two atomic oxygens from the iron-bound O2 into the indole 2 and 3 position. For establishment of the chemical mechanism of this unique enzymatic reaction, it is necessary to determine the conformation and electronic state of the substrate Trp bound to IDO. In this study, we measured the ultraviolet resonance Raman spectra of IDO in the presence of Trp to detect the vibrational modes of the substrate Trp. We compared the ultraviolet resonace Raman spectra of Trp in a ternary complex (Trp-bound cyanide enzyme) and a binary complex (Trp-bound reduced enzyme) of IDO with that of free Trp in solution and found that binding to IDO influences the conformation of Trp, resulting in similar changes in the two complexes, especially around the C3-C? bond. However, the presence of the diatomic ligand at the heme sixth coordination site in the ternary complex significantly alters the mobility and electronic structure of Trp, most likely resulting in the C2=C3 bond cleavage in the enzymatic reaction.

SUBMITTER: Yanagisawa S

PROVIDER: S-EPMC6712549 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1.

Yanagisawa Sachiko S Kayama Kure'e K Hara Masayuki M Sugimoto Hiroshi H Shiro Yoshitsugu Y Ogura Takashi T

Biophysical journal 20190719 4

Human indoleamine 2,3-dioxygenase 1 (IDO) is a heme enzyme that catalyzes the first reaction of the main metabolic pathway of L-tryptophan (Trp) to produce N-formylkynurenin. The reaction involves cleavage of the C2=C3 bond in the Trp indole ring and insertion of two atomic oxygens from the iron-bound O2 into the indole 2 and 3 position. For establishment of the chemical mechanism of this unique enzymatic reaction, it is necessary to determine the conformation an ...[more]

PMID: 31405517

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Project description:Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(•-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

Project description:Kynurenine (Kyn), a metabolite of tryptophan (Trp), is a key regulator of mammal immune responses such as cancer immune tolerance. Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) are main enzymes regulating the first and rate-limiting step of the Kyn pathway. To identify new small molecule inhibitors of TDO, we selected A172 glioblastoma cell line constitutively expressed TDO. Characterization of this cell line using kinase inhibitor library resulted in identification of MEK/ERK pathway-dependent TDO expression. After knowing the properties for TDO expression, we further proceeded to screen chemical library for TDO inhibitors. We previously determined that S-benzylisothiourea derivatives are enzymatic inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and suggested that the isothiourea moiety could be an important pharmacophore for binding to heme. Based on this premise, we screened an in-house library composed of various isothiourea derivatives and identified a bisisothiourea derivative, PVZB3001, as an inhibitor of TDO. Interestingly, PVZB3001 also inhibited the enzymatic activity of IDO1 in both cell-based and cell-free assays but did not inhibit other heme enzymes. Molecular docking studies suggested the importance of isothiourea moieties at the ortho position of the phenyl ring for the inhibition of catalytic activity. PVZB3001 showed competitive inhibition against TDO, and this was supported by the docking simulation. PVZB3001 recovered natural killer (NK) cell viability and functions by inhibiting Kyn accumulation in conditioned medium of both IDO1- and TDO-expressing cells. Furthermore, oral administration of IDO1-overexpressing tumor-bearing mice with PVZB3001 significantly inhibited tumor growth. Thus, we identified a novel selective dual inhibitor of IDO1 and TDO using the Kyn production assay with a glioblastoma cell line. This inhibitor could be a useful pharmacological tool for modulating the Kyn pathway in a variety of experimental systems.

Dataset Information

UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1.

Publications

UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1.

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