Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification

K Wilson,D.J Mole,M Binnie,N.Z.M Homer,X Zheng,B.A Yard,J.P Iredale,M Auer,S.P Webster

doi:10.1016/j.pep.2013.11.015

K Wilson, D.J Mole + Show 7 more

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https://doi.org/10.1016/j.pep.2013.11.015

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Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification.

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In mammals, the kynurenine pathway is the route of 99% of dietary tryptophan metabolism and is the primary route of synthesis of the essential cellular cofactor nicotinamide adenine dinucleotide (NAD)1 [1,2]
Expression of the full length kynurenine 3-monooxygenase (KMO) protein was apparent in the insoluble fraction as shown by the 55 kDa band on the SDS–PAGE gel (Fig. 3A)
In a kinetic assay measuring KMO activity in each fraction by the production of 3HK, KMO activity correlated with protein expression levels in soluble and insoluble fractions, with significant KMO enzymatic activity demonstrated in the insoluble pellet

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Introduction

The kynurenine pathway is the route of 99% of dietary tryptophan metabolism and is the primary route of synthesis of the essential cellular cofactor nicotinamide adenine dinucleotide (NAD)1 [1,2]. Central to the kynurenine pathway is the NADPH-dependant flavoprotein hydroxylase, kynurenine 3-monooxygenase (KMO) (EC 1.14.13.9) (Fig. 1). This enzyme catalyses the specific hydroxylation of kynurenine (L-Kyn) at the 3 position of its phenol ring to generate 3-hydroxykynurenine (3-HK) [3]. 3-HK, the product of KMO catalysis, exhibits toxicity to cells through reactive oxygen species generation, the cross-linking of proteins and mitochondrial respiratory chain inhibition [10]. KMO has recently been implicated as a therapeutic target for both Huntington’s disease [11] and post-traumatic sepsis [4]

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