首页|Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis

Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis

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  • Elsevier
Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD~+. Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD~+ levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihy-droxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.

Human NaPRTenzyme kineticshomology modelingmolecular dockingsite-directed mutagenesis

Lucia Galassi、Michele Di Stefano、Lucia Brunetti、Giuseppe Orsomando、Adolfo Amici、Silverio Ruggieri、Giulio Magni

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Dipartimento di Patologia Molecolare e Terapie Innovative - Sezione Biochimica, Universita Politecnica delle Marche, Via Ranieri 67, 60131 Ancona, Italy

Dipartimento di Patologia Molecolare e Terapie Innovative - Sezione Biochimica, Universita Politecnica delle Marche, Via Ranieri 67, 60131 Ancona, Italy,School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK

Dipartimento di Patologia Molecolare e Terapie Innovative - Sezione Biochimica, Universita Politecnica delle Marche, Via Ranieri 67, 60131 Ancona, Italy,Postdoctoral fellowship recipient co-funded by the Universita Politecnica delle Marche, Ancona, Italy

2012

10.1016/j.biochi.2011.06.033

Biochimie

Biochimie

SCI
ISSN：0300-9084
年,卷(期)：2012.94(2)
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