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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

Molecular docking of the hNaPRTase active site dimeric interface in complex with different ligands. Side chains of residues participating in the binding ligands ((A) Na; (B) NaMN; (C) Na and PRPP; (D) ATP) are represented as thin sticks and their identity is indicated, whereas ligands are depicted as green sticks. Hydrogen bonds are shown as yellow dotted lines.
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f0025: Molecular docking of the hNaPRTase active site dimeric interface in complex with different ligands. Side chains of residues participating in the binding ligands ((A) Na; (B) NaMN; (C) Na and PRPP; (D) ATP) are represented as thin sticks and their identity is indicated, whereas ligands are depicted as green sticks. Hydrogen bonds are shown as yellow dotted lines.

Mentions: Since we have been unable to obtain crystals of hNaPRTase in complex with any ligand, the binding mode of substrate/product to the enzyme active site was investigated through molecular docking simulations. The functional dimeric model of hNaPRTase was first re-built using the X-ray crystal structure and subsequently refined by molecular dynamics simulations. Na, NaMN, Na plus PRPP, and ATP were docked in the generated model. As already reported in the previously characterized NaPRTases [47], the active site consists of a pocket located at the dimer interface. In particular, in hNaPRTase it is delimited by the loops connecting α-helix 6 with β-strand 6 and α-helix 8 with β-strand 8 of α/β barrel domain of monomer A and completed by residues of β-strand 11 and α-helix 1 from monomer B (Fig. 4). According to our modeling procedure the pyridine ring of Na establishes contacts with the protein environment that involve a π–π stacking interaction with Y21B and Van der Walls contacts with L170A, G209A and L211A. In addition, the carboxyl group of Na is engaged in a strong ionic interaction with R318A (Fig. 5a). It has previously been reported that the G201 to Ala substitution causes a 90% reduction of enzyme activity even in the presence of ATP and that the Y21 to Ala substitution results in the complete loss of activity in absence of ATP and in a 50% reduction in the presence of ATP [28]. Our data are therefore in agreement with the functional analysis carried out by site directed mutagenesis and provide a solid structural explanation for the role played by these residues in catalysis. We then investigated the mode of binding for Na and PRPP when simultaneously bound to the enzyme active site (Fig. 5b). PRPP appears to be stabilized by two ionic interactions with R171A and K396B that are engaged in a salt bridge with the phosphate and pyrophosphate moieties, respectively. PRPP is further stabilized by two hydrogen bonds established between its ribose oxygen and R318A and between the pyrophosphate moiety and S214A. Our observations confirm the functional analysis carried out by site-directed mutagenesis previously reported in which the replacement of R318 with Ala completely abolish the enzyme activity in absence of ATP while a reduction of 50% is observed in the presence of ATP [28]. The ATP binding mode was also investigated and turned out to be quite similar to that observed for PRPP (Fig. 5d). In particular the ATP γ phosphate group is salt bridged to K396B, one of the key residue for PRPP binding, while the β phosphate group is involved in hydrogen bonds with H213A (backbone amide) and S214A. The adenine moiety of ATP is involved in a π-polar interaction with R318A. Also in this case, our observations remark an important role in catalysis for H213 that when mutated to Ala was reported to cause a 50% reduction of the enzyme activity [28]. Finally, the binding mode of the product NaMN was investigated (Fig. 5c). As expected, the orientation of both the Na and phosphate moieties in the mononucleotide, is identical to what observed for the corresponding chemical entities in the model of the Na-PRPP enzyme complex. In conclusion, our structural models for substrate binding provide a solid explanation for the role played in catalysis by residues that were previously reported to be essential for enzyme activity by a functional analysis based on site directed mutagenesis [28]. The perfect coherence between biochemical data reported in previous study and our structural observations, also proves that our molecular docking simulation are robust. Therefore, besides R318, Y21, H213 that were already subjected to site directed mutagenesis and that we confirm to be essential residues for catalysis, we propose that R171, K396 and S214 also play an essential role in substrate recognition. Moreover, we identify R318 as the major player in catalysis. Indeed, this residue is involved in the binding of all substrates by recognizing different chemical entities: the carboxylic moiety of Na, the ribose ring of PRPP and the adenine base of ATP. In addition, our in silico model of the complex hNaPRT-Na and hNaPRT-NaMN allows a detailed structural comparison of the mode of binding of Na, Nam and QA in the three phosphoribosyltransferases. At the level of the active sites we observe a high degree of structural conservation mainly between hNaPRTase and hNMPRTase, while a less significant conservation is observed with hQAPRTase. However, the mode of binding of all the three different pyridines is conserved, while different is the protein milieu surrounding the substrate. In particular, hQAPRTase provides a more positively charged environment that is required for the recognition of QA that carries two negatively charged carboxylic groups. Although our structural analysis does not disclose any obvious common features for the catalytic mechanism adopted by hQAPRTase, hNMPRTase and hNaPRTase, a conserved mode of binding of the pyridine ring containing substrate clearly emerges. More information can be extracted if the structural analysis is confined to hNaPRTase and hNMPRTase. In these two enzymes, the mode of binding of Na and Nam appears highly similar. Indeed, the pyridine ring is sandwiched by an aromatic/hydrophobic clamp in both enzymes: Y18/F193 in hNMPRTase and Y21/L170 in hNaPRTase. R311, R196 and K400 that in hNMPRTase play a key role in the binding of PRPP, are strictly conserved in hNaPRTase (R318, R171 and K396). On the other hand, a specific residue appears to participate in guaranteeing the strikingly substrate specificity displayed by hNMPRTase and hNAPRTase for nicotinamide versus nicotinic acid. While in hNMPRTase D219 interacts with the amide moiety of Nam, in hNaPRTase the structurally equivalent position is occupied by S214 contributing to direct the enzyme specificity toward nicotinate. Other structural determinants that do not clearly emerge in our docking models are however required to fully explain the strict specificity toward nicotinic acid displayed by hNaPRTase. Overall, a conserved mode of binding of QA, Na and Nam in the respective phosphoribosyltransferase is observed. However, the structural architecture of the active site is sensibly different in hQAPRTase and peculiar traits also feature hNMPRTase and hNaPRTase. Therefore, we propose that the wealth of structural information that is now available for all the three enzymes can be successfully exploited for the design of highly selective inhibitors.


Crystal structure of human nicotinic acid phosphoribosyltransferase.

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

Molecular docking of the hNaPRTase active site dimeric interface in complex with different ligands. Side chains of residues participating in the binding ligands ((A) Na; (B) NaMN; (C) Na and PRPP; (D) ATP) are represented as thin sticks and their identity is indicated, whereas ligands are depicted as green sticks. Hydrogen bonds are shown as yellow dotted lines.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0025: Molecular docking of the hNaPRTase active site dimeric interface in complex with different ligands. Side chains of residues participating in the binding ligands ((A) Na; (B) NaMN; (C) Na and PRPP; (D) ATP) are represented as thin sticks and their identity is indicated, whereas ligands are depicted as green sticks. Hydrogen bonds are shown as yellow dotted lines.
Mentions: Since we have been unable to obtain crystals of hNaPRTase in complex with any ligand, the binding mode of substrate/product to the enzyme active site was investigated through molecular docking simulations. The functional dimeric model of hNaPRTase was first re-built using the X-ray crystal structure and subsequently refined by molecular dynamics simulations. Na, NaMN, Na plus PRPP, and ATP were docked in the generated model. As already reported in the previously characterized NaPRTases [47], the active site consists of a pocket located at the dimer interface. In particular, in hNaPRTase it is delimited by the loops connecting α-helix 6 with β-strand 6 and α-helix 8 with β-strand 8 of α/β barrel domain of monomer A and completed by residues of β-strand 11 and α-helix 1 from monomer B (Fig. 4). According to our modeling procedure the pyridine ring of Na establishes contacts with the protein environment that involve a π–π stacking interaction with Y21B and Van der Walls contacts with L170A, G209A and L211A. In addition, the carboxyl group of Na is engaged in a strong ionic interaction with R318A (Fig. 5a). It has previously been reported that the G201 to Ala substitution causes a 90% reduction of enzyme activity even in the presence of ATP and that the Y21 to Ala substitution results in the complete loss of activity in absence of ATP and in a 50% reduction in the presence of ATP [28]. Our data are therefore in agreement with the functional analysis carried out by site directed mutagenesis and provide a solid structural explanation for the role played by these residues in catalysis. We then investigated the mode of binding for Na and PRPP when simultaneously bound to the enzyme active site (Fig. 5b). PRPP appears to be stabilized by two ionic interactions with R171A and K396B that are engaged in a salt bridge with the phosphate and pyrophosphate moieties, respectively. PRPP is further stabilized by two hydrogen bonds established between its ribose oxygen and R318A and between the pyrophosphate moiety and S214A. Our observations confirm the functional analysis carried out by site-directed mutagenesis previously reported in which the replacement of R318 with Ala completely abolish the enzyme activity in absence of ATP while a reduction of 50% is observed in the presence of ATP [28]. The ATP binding mode was also investigated and turned out to be quite similar to that observed for PRPP (Fig. 5d). In particular the ATP γ phosphate group is salt bridged to K396B, one of the key residue for PRPP binding, while the β phosphate group is involved in hydrogen bonds with H213A (backbone amide) and S214A. The adenine moiety of ATP is involved in a π-polar interaction with R318A. Also in this case, our observations remark an important role in catalysis for H213 that when mutated to Ala was reported to cause a 50% reduction of the enzyme activity [28]. Finally, the binding mode of the product NaMN was investigated (Fig. 5c). As expected, the orientation of both the Na and phosphate moieties in the mononucleotide, is identical to what observed for the corresponding chemical entities in the model of the Na-PRPP enzyme complex. In conclusion, our structural models for substrate binding provide a solid explanation for the role played in catalysis by residues that were previously reported to be essential for enzyme activity by a functional analysis based on site directed mutagenesis [28]. The perfect coherence between biochemical data reported in previous study and our structural observations, also proves that our molecular docking simulation are robust. Therefore, besides R318, Y21, H213 that were already subjected to site directed mutagenesis and that we confirm to be essential residues for catalysis, we propose that R171, K396 and S214 also play an essential role in substrate recognition. Moreover, we identify R318 as the major player in catalysis. Indeed, this residue is involved in the binding of all substrates by recognizing different chemical entities: the carboxylic moiety of Na, the ribose ring of PRPP and the adenine base of ATP. In addition, our in silico model of the complex hNaPRT-Na and hNaPRT-NaMN allows a detailed structural comparison of the mode of binding of Na, Nam and QA in the three phosphoribosyltransferases. At the level of the active sites we observe a high degree of structural conservation mainly between hNaPRTase and hNMPRTase, while a less significant conservation is observed with hQAPRTase. However, the mode of binding of all the three different pyridines is conserved, while different is the protein milieu surrounding the substrate. In particular, hQAPRTase provides a more positively charged environment that is required for the recognition of QA that carries two negatively charged carboxylic groups. Although our structural analysis does not disclose any obvious common features for the catalytic mechanism adopted by hQAPRTase, hNMPRTase and hNaPRTase, a conserved mode of binding of the pyridine ring containing substrate clearly emerges. More information can be extracted if the structural analysis is confined to hNaPRTase and hNMPRTase. In these two enzymes, the mode of binding of Na and Nam appears highly similar. Indeed, the pyridine ring is sandwiched by an aromatic/hydrophobic clamp in both enzymes: Y18/F193 in hNMPRTase and Y21/L170 in hNaPRTase. R311, R196 and K400 that in hNMPRTase play a key role in the binding of PRPP, are strictly conserved in hNaPRTase (R318, R171 and K396). On the other hand, a specific residue appears to participate in guaranteeing the strikingly substrate specificity displayed by hNMPRTase and hNAPRTase for nicotinamide versus nicotinic acid. While in hNMPRTase D219 interacts with the amide moiety of Nam, in hNaPRTase the structurally equivalent position is occupied by S214 contributing to direct the enzyme specificity toward nicotinate. Other structural determinants that do not clearly emerge in our docking models are however required to fully explain the strict specificity toward nicotinic acid displayed by hNaPRTase. Overall, a conserved mode of binding of QA, Na and Nam in the respective phosphoribosyltransferase is observed. However, the structural architecture of the active site is sensibly different in hQAPRTase and peculiar traits also feature hNMPRTase and hNaPRTase. Therefore, we propose that the wealth of structural information that is now available for all the three enzymes can be successfully exploited for the design of highly selective inhibitors.

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