title: Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis.
abstract: 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 dihydroxyacetone 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.
authors: Dipartimento di Patologia Molecolare e Terapie Innovative - Sezione Biochimica, Università Politecnica delle Marche, Via Ranieri 67, 60131 Ancona, Italy.
authors: Lucia
authors: L
authors: Galassi
authors: Michele
authors: M
authors: Di Stefano
authors: Lucia
authors: L
authors: Brunetti
authors: Giuseppe
authors: G
authors: Orsomando
authors: Adolfo
authors: A
authors: Amici
authors: Silverio
authors: S
authors: Ruggieri
authors: Giulio
authors: G
authors: Magni
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000096:biosynthesis*; D009525:Niacin / Q000378:metabolism*; D009536:Niacinamide / Q000378:metabolism; D009537:Nicotinamide Mononucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013403:Sugar Phosphates / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013403:Sugar Phosphates; D009243:NAD; D009537:Nicotinamide Mononucleotide; D009536:Niacinamide; D009525:Niacin; C002953:nicotinate mononucleotide; D000255:Adenosine Triphosphate; D010430:Pentosyltransferases; C021864:nicotinate phosphoribosyltransferase