The investigation of kynurenine 3-monooxygenase from Pseudomonas fluorescens: Mechanistic characterization, kinetic analyses, and inhibitor studies

Kynurenine 3-monoxygenase (KMO) is an NADPH-dependent flavoprotein hydroxylase that catalyzes the reaction of L-Kynurenine (L-Kyn) to 3-hydroxykynurenine (3OHKyn). The reaction is central to the tryptophan degradative pathway and takes place within microglial cells defining cellular concentrations of neurotoxin quinolinate and neuroprotectant kynurenate. Therefore KMO inhibition has been widely suggested as an early treatment for stroke and other neurological disorders that involve ischemia. Pseudomonas fluorescens str 17400, expresses five activities of tryptophan catabolism including that of KMO. The KMO gene from P. fluorescens was cloned and the protein was expressed and purified. It was determined that PfKMO was subject to inhibition by known mammalian KMO inhibitors and exhibited comparable ligand affinities for these molecules.;We have investigated the reductive and the oxidative half-reactions of KMO. The rate constant for reduction of the flavin cofactor by NADPH increases by a factor of ∼2.5x103 when L-Kyn is bound. KMO inhibitors whose structures are reminiscent of L-Kyn such as meta-nitrobenzoylalanine and benzoylalanine also stimulate flavin reduction by NADPH and, in the presence of dioxygen, result in the stoichiometric liberation of hydrogen peroxide, diminishing the perceived therapeutic potential of inhibitors of this type. This marks the importance of investigation of an enzyme's reaction mechanism instead of solely relying upon IC50 values or binding constants in the development of inhibitors. In the presence of the native substrate, the oxidative half-reaction exhibits triphasic absorbance data. Based on flavin difference spectra, the third phase can be correlated with product release. We have investigated the effects of pH, deuterium solvent, and ring-deuterated substrates on the oxidative half reaction of KMO and observed the contribution of multiple protons in flight during product release suggesting a dynamic conformational change on the enzyme that influences the spectrum of the flavin cofactor and shown definitively the existence of a non-aromatic form of the product that accumulates concomitant with hydroxylation.