Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase

Kim, Hyun Tae; Na, Byeong Kwan; Chung, Jiwoung; Kim, Sulhee; Kwon, Sool Ki; Cha, Hyunju; Son, Jonghyeon; Cho, Joong Myung; Hwang, Kwang Yeon

doi:10.1016/j.chembiol.2018.01.008

Cited by 26 publications

(34 citation statements)

References 72 publications

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“…Furthermore, regions of positive electrostatic potential in the active site would situate the anionic reduced flavin to the "in" position adjacent to the substrate, in addition to previously promoting the formation of the substrate phenolate [70]. Interestingly, a hydrogen-bonding network is not found in the active site of kynurenine 3-monooxygenase (KMO), in which conformational changes and flavin reduction are triggered by π-π interactions between the substrate and the loop above the re-side of the isoalloxazine group [71].…”

Section: Comparison Of Nahg With Others Flavoprotein Monooxygenasesmentioning

confidence: 99%

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase

Costa

Gómez

Araújo

et al. 2019

International Journal of Biological Macromolecules

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Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Å resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat /Km for the salicylate reaction catalyzed by the holo form is greater than 10 5 M -1 s -1 at pH 8.5 and 25 ºC.Hammett plots for Km and kcat using substituted salicylates indicates a change in rate-limiting step.Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.to the substrate phenol group [22]. In the reaction catalyzed by 3-hydroxybenzoate 6hydroxylase (3HB6H), which the substrate carboxylate group is meta to the phenol, the 3-hydroxybenzoate is converted to 2,5-dihydroxybenzoate through a hydroxylation followed by a deprotonation [23].Catalysis in these enzymes involves a C(4a)-hydroperoxyflavin species, which provides a powerful peroxo electrophile tuned for oxygen insertion at nucleophilic carbons and soft centers.Examples include hydroxylation coupled with different kinds of substitution, epoxidation, Baeyer-Villiger oxidation, and oxidation of heteroatoms (B, S, Se, N, and P) [24][25][26][27]. Formation of the C(4a)-hydroperoxyflavin species from the oxidized flavin and NAD(P)H requires two reactions coupled with dynamics of the isoalloxazine group between two positions [28]. In an external position, aside from the hydroxylation site, the oxidized isoaloxazine group is reduced by NAD(P)H. Then, the reduced isoaloxazine swings to an internal position, where it reacts with molecular oxygen to yield the C(4a)-hydroperoxyflavin species. This reaction is fast in enzymes, with rate constants typically between 10 4 and 10 6 M -1 s -1 at 4 ºC [29][30][31][32]; in contrast, the rate constant for the corresponding reaction in water is about 250 M -1 s -1 at 30 ºC [33].Herein, we explore the catalytic mechanism of NahG, that, like those other monooxygenases, remains a subject of intense debate [10,20]. Particular goals are to identify the catalytic groups involved in catalysis, the reactivity of reaction intermediates, and the reaction pathway that affords the products. The mechanistic proposal resulting from this work is supported by kinetics and x-ray crystallogra...

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Section: Comparison Of Nahg With Others Flavoprotein Monooxygenasesmentioning

confidence: 99%

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase

Costa

Gómez

Araújo

et al. 2019

International Journal of Biological Macromolecules

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“…Studies on the structure and mechanism of KMO in animal (Zhang et al, ) have been discussed in the past (Kim et al, ; Smith et al, ). KMO is situated in the outer membrane of mitochondria (Quan et al, ) as a membrane‐associated protein (Gao et al, ), and its crystal structure was found by Amaral et al () as well as the first successful bacterial ( Escherichia coli ) expression of active human KMO enzyme expressed in the soluble fraction found by Wilson et al ().…”

Section: The Structure and Function Of Kmomentioning

confidence: 99%

Kynurenine‐3‐monooxygenase: A new direction for the treatment in different diseases

Lü

Shao

2020

Food Science & Nutrition

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Kynurenine‐3‐monooxygenase (KMO) is an enzyme that relies on nicotinamide adenine dinucleotide phosphate (NADP), a key site in the kynurenine pathway (KP), which has great effects on neurological diseases, cancer, and peripheral inflammation. This review mainly pay attention to the research of KMO mechanism for the treatment of different diseases, and hopes to provide assistance for clinical and drug use. KMO controlling the chief division of the KP, which directly controls downstream product quinolinic acid (QUIN) and indirectly controls kynurenic acid (KYNA), plays an important role in many diseases, especially neurological diseases.

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“…The enzyme from P. fluorescens (PfKMO) is a soluble enzyme with 37% identity to human KMO that can be expressed heterologously in Escherichia coli (Crozier and Moran, 2007). The crystal structures of PfKMO with a number of inhibitors and L-kynurenine bound have been solved recently (Hutchinson et al, 2017; Gao et al, 2018; Kim et al, 2018). The structure of PfKMO (Figure 3) is very similar to that of ScKMO.…”

Section: The Three Dimensional Structure Of Kynurenine Monooxygenase mentioning

confidence: 99%

“…The first structure of hKMO was reported recently (Kim et al, 2018). The crystal structure was solved to a resolution of 2.1 Å after engineering the deletion mutant, hKMO-374 (residues 1-374), in which the transmembrane domains were deleted, in order to obtain a human KMO protein suitable for crystallization.…”

Section: The Three Dimensional Structure Of Kynurenine Monooxygenase mentioning

confidence: 99%

“…In the presence of m -NBA, and UPF-648, there is an accumulation of the hydroperoxyflavin, which decays to yield hydrogen peroxide. A recent study provided a better understanding on how the uncoupling happens (Kim et al, 2018). The authors proposed the flavin reduction in KMO is associated with conformational changes, via π- π interactions between a substrate or an NADPH uncoupler, and the loop above the re- side of the flavin.…”

Section: Kmo Inhibitorsmentioning

confidence: 99%

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Modulation of Enzyme Activity in the Kynurenine Pathway by Kynurenine Monooxygenase Inhibition

Phillips

Iradukunda

Hughes

et al. 2019

Front. Mol. Biosci.

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The kynurenine pathway is the major route for tryptophan metabolism in mammals. Several of the metabolites in the kynurenine pathway, however, are potentially toxic, particularly 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and quinolinic acid. Quinolinic acid (QUIN) is an excitotoxic agonist at the NMDA receptor, and has been shown to be elevated in neurodegenerative diseases such as Alzheimer's Disease and Huntington's Disease. Thus, inhibitors of enzymes in the kynurenine pathway may be valuable to treat these diseases. Kynurenine monooxygenase (KMO) is the ideal target for an inhibitor, since inhibition of it would be expected to decrease the toxic metabolites and increase kynurenic acid (KynA), which is neuroprotective. The first generation of KMO inhibitors was based on structural analogs of the substrate, L-kynurenine. These compounds showed reduction of QUIN and increased KynA in vivo in rats. After the determination of the x-ray crystal structure of yeast KMO, inhibitor design has been facilitated. Benzisoxazoles with sub-nM binding to KMO have been developed recently. Some KMO ligands promote the reaction of NADPH with O2 without hydroxylation, resulting in uncoupled formation of H2O2. This potentially toxic side reaction should be avoided in the design of drugs targeting the kynurenine pathway for treatment of neurodegenerative disorders.

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Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase

Cited by 26 publications

References 72 publications

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase

Kynurenine‐3‐monooxygenase: A new direction for the treatment in different diseases

Modulation of Enzyme Activity in the Kynurenine Pathway by Kynurenine Monooxygenase Inhibition

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