Structural and kinetic studies of the NADPH dependent oxidoreductases kynurenine 3-monooxygenase and light-dependent protochlorophyllide oxidoreductase

Abstract

NADPH-dependent oxidoreductases catalyse around 25% of all reactions within the oxidoreductase family of enzymes. They are essential in many biological reactions and play a crucial role in cell metabolism. Studies of NADPH-dependent oxidoreductases can help us gain insights into their biological roles, and can also promote their applications in chemical and pharmaceutical industries. In the present work two NADPH-dependent oxidoreductases, kynurenine 3-monooxygenase (KMO) and light-dependent protochlorophyllide oxidoreductase (POR), have been studied by a combination of structural and kinetic measurements. KMO lies at a branching point of the kynurenine pathway (KP) in tryptophan metabolism, the dysregulation of which will result in the imbalances of neuroactive metabolites. As the KP has been implicated in several neurological diseases, KMO is a promising drug target for these disorders. Through virtual screening, we have identified and characterised 19 KMO inhibitors. By using a prodrug strategy to modify one of these inhibitors, a new prodrug variant was produced that is able to cross the blood brain barrier and considerably decrease the toxic KP metabolite 3-hydoxykynurenine in the brain. The prodrug will advance the development of therapeutic strategies for KP related neurodegenerative and neuroinflammatory diseases. Light-dependent POR catalyses the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a crucial reaction in chlorophyll biosynthesis. The lack of a 3D structure has meant that a detailed molecular understanding of the mechanism has been hampered for many years. Herein, we solved the crystal structure of Thermosynechococcus elongatus POR in its apo form and in complex with the nicotinamide coenzyme. A ternary complex model generated by molecular docking and molecular dynamics simulations was validated by biophysical studies and provided a structural basis for POR photocatalysis. Further investigation of lid like regions, lid1 and lid2, which cover the Pchlide molecule in the active site enabled us to obtain a better understanding of the role of these regions in the POR reaction. Lid1 varies significantly between cyanobacteria and plants and influences the proton transfer step by perturbing the proton relay channel around the POR active site. Lid2 is a highly conserved hydrophobic region that appears to be crucial for the optimal positioning of the Pchlide molecule in the active site for efficient photochemistry. The work shows how the POR active site is highly tuned to facilitate photocatalysis.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nigel Scrutton (Supervisor) & Sam Hay (Supervisor)