Structural Insights into the Quaternary Catalytic Mechanism of Hexameric Human Quinolinate Phosphoribosyltransferase, a Key Enzyme in de novo NAD Biosynthesis.

Young Jin Lee,Tae Gyun Kim,Kyoung Ryoung Park,Jun Hyuck Lee,Jung Youn Kang,Soo Hyun Eom,Hyung-Seop Youn,Gil Bu Kang,Young Jun Im,Jun Yop An,Jung-Gyu Lee,Mun-Kyoung Kim

doi:10.1038/srep19681

Young Jin Lee, Tae Gyun Kim + Show 10 more

Open Access

https://doi.org/10.1038/srep19681

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Abstract

Quinolinate phosphoribosyltransferase (QPRT) catalyses the production of nicotinic acid mononucleotide, a precursor of de novo biosynthesis of the ubiquitous coenzyme nicotinamide adenine dinucleotide. QPRT is also essential for maintaining the homeostasis of quinolinic acid in the brain, a possible neurotoxin causing various neurodegenerative diseases. Although QPRT has been extensively analysed, the molecular basis of the reaction catalysed by human QPRT remains unclear. Here, we present the crystal structures of hexameric human QPRT in the apo form and its complexes with reactant or product. We found that the interaction between dimeric subunits was dramatically altered during the reaction process by conformational changes of two flexible loops in the active site at the dimer-dimer interface. In addition, the N-terminal short helix α1 was identified as a critical hexamer stabilizer. The structural features, size distribution, heat aggregation and ITC studies of the full-length enzyme and the enzyme lacking helix α1 strongly suggest that human QPRT acts as a hexamer for cooperative reactant binding via three dimeric subunits and maintaining stability. Based on our comparison of human QPRT structures in the apo and complex forms, we propose a drug design strategy targeting malignant glioma.

Highlights

Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, exists in both oxidised (NAD+, electron acceptor) and reduced (NADH, electron donor) forms
To investigate the molecular basis of mammalian Quinolinate phosphoribosyltransferase (QPRT) in the hexamer state upon substrate binding, we determined the structures of HsQPRT in the apo form and in complex with its reactant quinolinic acid (QA) as well as the reaction product NAMN at 2.8, 3.1 and 2.6 Å, respectively
The HsQPRT monomer consists of 12 α -helices and 12 β -strands arranged into two structural domains: an N-terminal open-faced α /β -sandwich domain and a C-terminal open TIM α /β -barrel domain (Fig. 1a)

Summary

Introduction

Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, exists in both oxidised (NAD+, electron acceptor) and reduced (NADH, electron donor) forms. Most prokaryotic QPRTs exist as dimers, the enzymes from Helicobacter pylori and Thermus thermophilus have a hexameric structure that is identical to HsQPRT-tartrate and ScQPRT23,24,26,27. Numerous structures of HsQPRT have been determined in one apo form and two complexes with reactant QA analogues (tartrate and phthalate), the molecular basis for the existence of HsQPRT and the catalysis of the phosphoribosyl transfer reaction as a hexamer remains unclear. Comparison of the apo enzyme structures reveal that, the reactant (QA) and the reaction product (NAMN) complexes have a conformational change that dramatically affected two distinctive flexible loops around the active site as well as the interface forming the hexamer, indicating that the reaction mediated by hexameric HsQPRT is regulated by the interaction between two dimeric subunits. Analysis of the range of structural diversity of QPRTs has clinical implications in the development of anticancer agents in the treatment of malignant glioma

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