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Crystal structure of human nicotinic acid phosphoribosyltransferase.

Marletta AS, Massarotti A, Orsomando G, Magni G, Rizzi M, Garavaglia S - FEBS Open Bio (2015)

Bottom Line: Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers.The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism.Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy.

ABSTRACT
Nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss-Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9 Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent.

No MeSH data available.


Related in: MedlinePlus

FK866-binding-site: structural comparison between hNMPRTase and hNaPRTase. Human NMPRTase structure in complex with its inhibitor FK866 (PDB ID: 2GVJ) is colored in gray and hNaPRTase is colored in orange. Structural superposition between these two structures highlight, in hNaPRTase, of the tunnel where FK866 binds in hNMPRTase. Buried surface areas of hNMPRTase (upper left panel) and hNaPRTase (lower left panel) monomers are represented and FK866 is shown as green stick. A ribbon represented close-up view (right panel) of the superposed secondary structure elements sterically hampering (in hNaPRTase) or permitting (in hNMPRTase) the binding of FK866. Steric hindrance of FK866 is represented by light green spheres.
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f0035: FK866-binding-site: structural comparison between hNMPRTase and hNaPRTase. Human NMPRTase structure in complex with its inhibitor FK866 (PDB ID: 2GVJ) is colored in gray and hNaPRTase is colored in orange. Structural superposition between these two structures highlight, in hNaPRTase, of the tunnel where FK866 binds in hNMPRTase. Buried surface areas of hNMPRTase (upper left panel) and hNaPRTase (lower left panel) monomers are represented and FK866 is shown as green stick. A ribbon represented close-up view (right panel) of the superposed secondary structure elements sterically hampering (in hNaPRTase) or permitting (in hNMPRTase) the binding of FK866. Steric hindrance of FK866 is represented by light green spheres.

Mentions: FK866 is a potent hNMPRTase competitive inhibitor endowed with a strong antitumor activity and is currently undergoing clinical trials for the treatment of cancer [31]. Surprisingly, FK866 does not inhibit hNaPRTase [31] and an explanation of this unexpected observation is still missing. The inhibitory mechanism of action on the target has been investigated by structural studies [31]. FK866 binds to hNMPRTase in a tunnel observed at the dimer interface and defined by the parallel β sheet of the α/β barrel domain of each monomer [31]. In particular, two small β strands (β14 and β15) that participate in shaping the tunnel and are located in close proximity of the enzyme’s active site, contribute to the recognition and binding of FK866. In hNaPRTase the corresponding region (β13 and β14) revealed a different orientation determined by a 10 residues deletion that shorten the preceding alpha helix (α14). The resulting structural arrangement sterically prevents the binding of FK866 to hNaPRTase due to severe clashes between the inhibitor and the protein region defining the tunnel (Fig. 7).


Crystal structure of human nicotinic acid phosphoribosyltransferase.

Marletta AS, Massarotti A, Orsomando G, Magni G, Rizzi M, Garavaglia S - FEBS Open Bio (2015)

FK866-binding-site: structural comparison between hNMPRTase and hNaPRTase. Human NMPRTase structure in complex with its inhibitor FK866 (PDB ID: 2GVJ) is colored in gray and hNaPRTase is colored in orange. Structural superposition between these two structures highlight, in hNaPRTase, of the tunnel where FK866 binds in hNMPRTase. Buried surface areas of hNMPRTase (upper left panel) and hNaPRTase (lower left panel) monomers are represented and FK866 is shown as green stick. A ribbon represented close-up view (right panel) of the superposed secondary structure elements sterically hampering (in hNaPRTase) or permitting (in hNMPRTase) the binding of FK866. Steric hindrance of FK866 is represented by light green spheres.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4442680&req=5

f0035: FK866-binding-site: structural comparison between hNMPRTase and hNaPRTase. Human NMPRTase structure in complex with its inhibitor FK866 (PDB ID: 2GVJ) is colored in gray and hNaPRTase is colored in orange. Structural superposition between these two structures highlight, in hNaPRTase, of the tunnel where FK866 binds in hNMPRTase. Buried surface areas of hNMPRTase (upper left panel) and hNaPRTase (lower left panel) monomers are represented and FK866 is shown as green stick. A ribbon represented close-up view (right panel) of the superposed secondary structure elements sterically hampering (in hNaPRTase) or permitting (in hNMPRTase) the binding of FK866. Steric hindrance of FK866 is represented by light green spheres.
Mentions: FK866 is a potent hNMPRTase competitive inhibitor endowed with a strong antitumor activity and is currently undergoing clinical trials for the treatment of cancer [31]. Surprisingly, FK866 does not inhibit hNaPRTase [31] and an explanation of this unexpected observation is still missing. The inhibitory mechanism of action on the target has been investigated by structural studies [31]. FK866 binds to hNMPRTase in a tunnel observed at the dimer interface and defined by the parallel β sheet of the α/β barrel domain of each monomer [31]. In particular, two small β strands (β14 and β15) that participate in shaping the tunnel and are located in close proximity of the enzyme’s active site, contribute to the recognition and binding of FK866. In hNaPRTase the corresponding region (β13 and β14) revealed a different orientation determined by a 10 residues deletion that shorten the preceding alpha helix (α14). The resulting structural arrangement sterically prevents the binding of FK866 to hNaPRTase due to severe clashes between the inhibitor and the protein region defining the tunnel (Fig. 7).

Bottom Line: Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers.The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism.Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy.

ABSTRACT
Nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss-Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9 Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent.

No MeSH data available.


Related in: MedlinePlus