Functional characterization and mechanism of action of recombinant human kynurenine 3‐hydroxylase

Abstract

The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol´min 21´m g 21 . Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl 2 , and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.

Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

“…A soluble, bacterial form of KMO from Pseudomonas fluorescens has been expressed efficiently in Escherichia coli 20 ; however, our attempts at expressing the full-length human protein in this host were not successful. Recombinant human KMO has been expressed in mammalian cells by transient transfection, 10,21 but the low levels of expression reported in these systems would have been prohibitive for an extended screening campaign. At the outset of this work, an HEK293 stable cell line expressing full-length human KMO, previously generated for cell-based assay purposes, was evaluated.…”

“…8 Studies have already shown positive effects of KMO inhibitors in brain injury models, 9 although poor penetration of the blood-brain barrier is problematic. 7 Previously described assays for KMO, such as the measurement of absorbance of NADPH, 10 are typically insensitive and prone to interference by contaminating enzyme activities. A radiometric assay, based on the release of tritiated water, was described by Erickson et al 11 and has been adapted to a 96-well plate format 12 but is unsuited to largescale automation.…”

Enzymology of NAD+ homeostasis in man

“…The observation that 3-hydroxykynurenine induces an H 2 O 2 -mediated neurotoxicity in the central nervous system [62,63] has stimulated extensive research on this enzyme. Indeed, K3H represents an attractive pharmacological target in that its inhibition both prevents the formation of a neurotoxic molecule and fosters the production of kynurenic acid, a known neuroprotective metabolite [64], through a divergent route catalyzed by kynurenine aminotransferase [65]. This strongly stimulates the search for novel inhibitors of the enzyme [66].…”

“…This strongly stimulates the search for novel inhibitors of the enzyme [66]. The human K3H cDNA has been isolated and expressed both in human embryonic kidney fibroblasts (HEK-293) and COS-1 cells [58,65]. The corresponding gene is localized to chromosome 1q43 [67].…”

The tryptophan oxidation pathway in mosquitoes with emphasis on xanthurenic acid biosynthesis

“…The catalytic mechanism of KMO has been suggested as being similar to that of a bacterial phydroxybenzoate hydroxylase (Entsch et al, 1976a,b;Breton et al, 2000). Sequence comparison between the Ae.…”