ENZYMATIC SYSTEMS AND METHODS FOR SYNTHESIZING NICOTINAMIDE MONONUCLEOTIDE AND NICOTINIC ACID MONONUCLEOTIDE

Abstract

Enzyme-based systems and methods for synthesizing the NAD precursors NMN and NaMN are disclosed. Such methods and systems utilize a mutated form of phosphoribosylpyrophosphate synthetase (PRS) that is superactive and/or other enzyme or enzyme combinations that are immobilized onto a solid surface. The methods and systems substantially increase the efficiency and yield of NAD precursor synthesis.

Claims

1. A system for synthesizing a nicotinamide adenine dinucleotide (NAD) precursor, the system comprising a superactive phosphoribosylpyrophosphate synthetase (PRS) mutant, wherein the PRS mutant is less sensitive to the product of the reaction that it catalyzes than a wild type PRS.

2. The system of claim 1, wherein the superactive PRS mutant comprises a polypeptide that differs from wild type PRS by one or more amino acid substitutions.

3. The system of claim 1 or claim 2, wherein the one or more amino acid substitutions are selected from the group consisting of Asp51His of human PRS, Asn113Ser of human PRS, Leu128Ile of human PRPP, Asp182His of human PRS, Ala189Val of human PRS, His192Gln of human PRS, any of the equivalent substitutions in a non-human PRS, and any combination thereof.

4. The system of any of claims 1-3, wherein the superactive PRS mutant comprises one or more affinity tags.

5. The system of claim 4, wherein the affinity tag is a 6xHis tag or a glutathione S-transferase (GST) tag.

6. The system of any of claims 1-5, wherein the superactive PRS mutant is recombinantly produced, isolated, or purified.

7. The system of any of claims 1-6, wherein the superactive PRS mutant is immobilized onto a surface.

8. The system of claim 7, wherein the superactive PRS mutant is immobilized onto the surface by adsorption, affinity binding, ionic bonding, or covalent bonding.

9. The system of claim 7 or claim 8, wherein the surface is the surface of a bead or comprises a resin.

10. The system of any of claims 1-9, further comprising nicotinamide phosphoribosyltransferase (NAMPT) or nicotinate phosphoribosyltransferase (NAPRT).

11. The system of claim 10, wherein the NAMPT or NAPRT comprises one or more affinity tags.

12. The system of claim 11, wherein the affinity tag is a 6xHis tag or a glutathione S-transferase (GST) tag.

13. The system of any of claims 10-13, wherein the NAMPT or NAPRT is recombinantly produced, isolated, or purified.

14. The system of any of claims 10-13, wherein the NAMPT or NAPRT is immobilized onto a surface.

15. The system of claim 14, wherein the NAMPT or NAPRT is immobilized onto the surface by adsorption, affinity binding, ionic bonding, or covalent bonding.

16. The system of claim 14 or claim 15, wherein the surface is the surface of a bead or comprises a resin.

17. The system of any of claims 14-16, wherein, the PRS mutant is also immobilized onto a surface.

18. The system of claim 17, wherein the PRS mutant and the NAMPT or NAPRT are immobilized onto different surfaces.

19. The system of claim 17, wherein the PRS mutant and the NAMPT or NAPRT are immobilized onto the same surface.

20. The system of any of claim 1-19, further comprising adenosine triphosphate (ATP).

21. The system of any of claims 1-20, further comprising ribose-5-phosphate.

22. The system of any of claims 1-21, further comprising nicotinamide or nicotinic acid.

23. The system of any of claims 1-22, further comprising phosphoribosyl pyrophosphate (PRPP).

24. The system of any of claims 1-23, further comprising nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN).

25. A method for synthesizing an nicotinamide adenine dinucleotide (NAD) precursor comprising contacting ribose-5-phosphate with a superactive phosphoribosylpyrophosphate synthetase (PRS) mutant in the presence of adenosine triphosphate (ATP), wherein the PRS mutant is less sensitive to the product of the reaction that it catalyzes than a wild type PRS, and whereby phosphoribosyl pyrophosphate (PRPP) is produced.

26. The method of claim 25, wherein the superactive PRS mutant comprises a polypeptide that differs from wild type PRS by one or more amino acid substitutions.

27. The method of claim 25 or claim 26, wherein the one or more amino acid substitutions are selected from the group consisting of Asp51His of human PRS, Asn113Ser of human PRS, Leu128Ile of human PRPP, Asp182His of human PRS, Ala189Val of human PRS, His192Gln of human PRS, any of the equivalent substitutions in a non-human PRS, and any combination thereof.

28. The method of any of claims 25-27, wherein the superactive PRS mutant comprises one or more affinity tags.

29. The method of claim 28, wherein the affinity tag is a 6xHis tag or a glutathione S-transferase (GST) tag.

30. The method of any of claims 25-29, wherein the superactive PRS mutant is recombinantly produced, isolated, or purified.

31. The method of any of claims 25-30, wherein the superactive PRS mutant is immobilized onto a surface.

32. The method of claim 31, wherein the superactive PRS mutant is immobilized onto the surface by adsorption, affinity binding, ionic bonding, or covalent bonding.

33. The method of claim 31 or claim 32, wherein the surface is the surface of a bead or comprises a resin.

34. The method of any of claims 25-33, further comprising: (a) contacting the resulting PRPP with nicotinamide phosphoribosyltransferase (NAMPT) in the presence of nicotinamide, whereby nicotinamide monocleotide (NMN) is produced; or (b) contacting the resulting PRPP with nicotinate phosphoribosyltransferase (NAPRT) in the presence of nicotinic acid, whereby nicotinic acid mononucleotide (NaMN) is produced.

35. The method of claim 34, wherein the NAMPT or NAPRT comprises one or more affinity tags.

36. The method of claim 35, wherein the affinity tag is a 6xHis tag or a glutathione S-transferase (GST) tag.

37. The method of any of claims 34-36, wherein the NAMPT or NAPRT is recombinantly produced, isolated, or purified.

38. The method of any of claims 34-37, wherein the NAMPT or NAPRT is immobilized onto a surface.

39. The method of claim 38, wherein the NAMPT or NAPRT is immobilized onto the surface by adsorption, affinity binding, ionic bonding, or covalent bonding.

40. The method of claim 38 or claim 39, wherein the surface is the surface of a bead or comprises a resin.

41. The method of any of claims 38-40, wherein, the PRS mutant is also immobilized onto a surface.

42. The method of claim 41, wherein the PRS mutant and the NAMPT or NAPRT are immobilized onto different surfaces.

43. The method of claim 17, wherein the PRS mutant and the NAMPT or NAPRT are immobilized onto the same surface.

44. The method of any of claims 34-43, further comprising purifying or concentrating the NMN or NaMN produced.

45. A system for synthesizing nicotinamide mononucleotide (NMN), the system comprising nicotinamide riboside kinase (NRK) immobilized onto a surface.

46. The system of claim 45, wherein the NRK comprises one or more affinity tags.

47. The system of claim 46, wherein the affinity tag is a 6xHis tag or a glutathione S-transferase (GST) tag.

48. The system of any of claims 45-47, wherein the NRK is recombinantly produced, isolated, or purified.

49. The system of any of claims 45-48, wherein the NRK is immobilized onto the surface by adsorption, affinity binding, ionic bonding, or covalent bonding.

50. The system of any of claims 45-49, wherein the surface is the surface of a bead or comprises a resin.

51. the system of any of claims 45-50, wherein the NRK is purified from cells or produced through recombinant means.

52. The system of any of claims 45-51, further comprising adenosine triphosphate (ATP).

53. The system of any of claims 45-52, further comprising nicotinamide riboside.

54. The system of any of claims 45-53, further comprising nicotinamide mononucleotide (NMN).

55. A method for synthesizing nicotinamide mononucleotide (NMN), the method comprising contacting nicotinamide riboside kinase (NRK) immobilized onto a surface with nicotinamide riboside in the presence of adenosine triphosphate (ATP), whereby NMN is produced.

56. The method of claim 55, wherein the NRK comprises one or more affinity tags.

57. The method of claim 56, wherein the affinity tag is a 6xHis tag or a glutathione S-transferase (GST) tag.

58. The method of any of claims 55-57, wherein the NRK is recombinantly produced, isolated, or purified.

59. The method of any of claims 55-58, wherein the NRK is immobilized onto the surface by adsorption, affinity binding, ionic bonding, or covalent bonding.

60. The method of any of claims 55-59, wherein the surface is the surface of a bead or comprises a resin.

61. The method of any of claims 55-60, further comprising purifying or concentrating the NMN produced.

62. A system for synthesizing nicotinamide mononucleotide (NMN), the system comprising the following enzymes immobilized onto a surface: (a) a superactive phosphoribosylpyrophosphate synthetase (PRS) mutant, wherein the PRS mutant is less sensitive to the product of the reaction that it catalyzes than a wild type PRS; (b) hexokinase; (c) glucose-6phosphate dehydrogenase; (d) gluconolactonase; (e) 6-phospho gluconate dehydrogenase; (f) ribulose-5-phosphate isomerase; and (g) nicotinamide phosphoribosyl transferase.

63. The system of claim 62, wherein one or more of the immobilized enzymes comprises one or more affinity tags.

64. The system of claim 63, wherein the affinity tag is a 6xHis tag or a glutathione S-transferase (GST) tag.

65. The system of any of claims 62-64, wherein one or more of the immobilized enzymes is recombinantly produced, isolated, or purified from a cell.

66. The system of any of claims 62-65, wherein the NRK is immobilized onto the surface by adsorption, affinity binding, ionic bonding, or covalent bonding.

67. The system of any of claims 62-66, wherein the superactive PRS mutant comprises a polypeptide that differs from wild type PRS by one or more amino acid substitutions.

68. The system of claim 67, wherein the one or more amino acid substitutions are selected from the group consisting of Asp51His of human PRS, Asn113Ser of human PRS, Leu128Ile of human PRPP, Asp182His of human PRS, Ala189Val of human PRS, His192Gln of human PRS, any of the equivalent substitutions in a non-human PRS, and any combination thereof.

69. The system of any of claims 62-68, wherein the surface is the surface of a bead or comprises a resin.

70. The system of any of claims 62-69, wherein each enzyme is immobilized onto a different surface.

71. The system of any of claims 62-69, wherein each enzyme is immobilized onto a different surface.

72. The system of any of claims 62-69, wherein the six immobilized enzymes are immobilized to between two and five different surfaces.

73. The system of any of claims 62-72, further comprising one or more of the group consisting of glucose, nicotinamide, adenosine triphosphate (ATP), Nicotinamide adenine dinucleotide phosphate (NADP.sup.+), an oxidizing agent, and mixtures thereof.

74. A method for synthesizing nicotinamide mononucleotide (NMN), the method comprising contacting the system of any of claims 62-72 with nicotinamide in the presence of glucose, adenosine triphosphate (ATP), Nicotinamide adenine dinucleotide phosphate (NADP.sup.+), and an oxidizing agent; whereby NMN is produced.

75. The method of claim 74, further comprising purifying or concentrating the NMN produced.

Description

DETAILED DESCRIPTION

I. Definitions

[0058] The phrase a or an entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms a (or an), one or more, and at least one can be used interchangeably herein.

[0059] The terms optional or optionally as used herein means that a subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, optional bond means that the bond may or may not be present, and that the description includes single, double, or triple bonds.

[0060] The term purified, as described herein, refers to the purity of a given compound. For example, a compound is purified when the given compound is a major component of the composition, i.e., at least 50% w/w pure. Thus, purified embraces at least 50% w/w purity, at least 60% w/w purity, at least 70% purity, at least 80% purity, at least 85% purity, at least 90% purity, at least 92% purity, at least 94% purity, at least 96% purity, at least 97% purity, at least 98% purity, at least 99% purity, at least 99.5% purity, and at least 99.9% purity, wherein substantially pure embraces at least 97% purity, at least 98% purity, at least 99% purity, at least 99.5% purity, and at least 99.9% purity.

[0061] Nicotinamide, which corresponds to the following structure,

##STR00001##

is one of the two principal forms of the B-complex vitamin niacin. The other principal form of niacin is nicotinic acid; nicotinamide, rather than nicotinic acid, however, is the major substrate for nicotinamide adenine dinucleotide (NAD) biosynthesis in mammals, as discussed in detail herein. Nicotinamide, in addition to being known as niacinamide, is also known as 3-pyridinecarboxamide, pyridine-3-carboxamide, nicotinic acid amide, vitamin B3, and vitamin PP. Nicotinamide has a molecular formula of C.sub.6H.sub.6N.sub.2O and its molecular weight is 122.13 Daltons. Nicotinamide is commercially available from a variety of sources.

[0062] Nicotinamide Adenine Dinucleotide (NAD.sup.+), which corresponds to the following structure,

##STR00002##

is produced from the conversion of nicotinamide to NMN, which is catalyzed by Nampt, and the subsequent conversion of NMN to NAD, which is catalyzed by Nmnat. Nicotinamide adenine dinucleotide (NAD) has a molecular formula of C.sub.21H.sub.27N.sub.7O.sub.14P.sub.2 and a molecular weight of 663.43. Nicotinamide adenine dinucleotide (NAD) is commercially available from such sources as Sigma-Aldrich (St. Louis, Mo.).

[0063] Nicotinamide Mononucleotide (NMN), which corresponds to the following structure,

##STR00003##

is produced from nicotinamide in the NAD biosynthesis pathway, a reaction that is catalyzed by Nampt. NMN is further converted to NAD in the NAD biosynthesis pathway, a reaction that is catalyzed by Nmnat. Nicotinamide mononucleotide (NMN) has a molecular formula of C.sub.11H.sub.15N.sub.2O.sub.8P and a molecular weight of 334.22. Nicotinamide mononucleotide (NMN) is commercially available from such sources as Sigma-Aldrich (St. Louis, Mo.).

[0064] Nicotinamide Riboside (NR), which corresponds to the following structure,

##STR00004##

is characterized and a synthesized as described in, for instance, U.S. Pat. No. 8,106,184.

[0065] Nicotinic Acid Mononucleotide (NaMN) corresponds to the following structure:

##STR00005##

[0066] Nicotinic Acid Riboside (NaR) corresponds to the following structure:

##STR00006##

II. Description of Selected Exemplary Embodiments

[0067] The following exemplary methods and systems are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following exemplary systems and methods.

Exemplary Method 1

[0068] The human enzyme phosphoribosoylpyrophosphate synthetase (PRS) is mutated to increase its activity through rendering it insensitive to the product of its own reaction, phosphoribosyl pyrophosphate (PRPP). Mutations may include, without limitation, Asp51His, Asn113Ser, Leu128Ile, Asp182His, Ala189Val and His192Gln. These mutations are defined relative to the known sequence of human PRS. However, PRS from other species may be used in the disclosed systems and methods, with equivalent mutations in non-human homologs also resulting in the required superactivity.

[0069] Enzymes may optionally be tagged with affinity tags, such as 6xHis tag or GST tag. Recombinant or purified enzyme mutants as described above may be immobilized on, for example, beads or resin (e.g., agarose beads, sepharose beads) through adsorption, affinity binding (e.g. 6xHis tagged proteins to Ni.sup.2+ or Co.sup.2+ beads), ionic binding or covalent bonds. Such methods are well-known in the art.

[0070] Ribose-5-phosphate in the presence of ATP may be passed through beads or resin with immobilized, mutated PRS to yield PRPP. The enzyme nicotinamide phosphoribosyltransferase (NAMPT) may be tagged and immobilized to beads or resin, as described above and known in the art. The product of the previous reaction can be combined with nicotinamide and passed through such a resin to yield nicotinamide mononucleotide (NMN).

[0071] In an alternative method, resin or beads carrying recombinant or isolated NAMPT are placed in a bottom layer of a column, and resin or beads carrying recombinant or isolated PRS mutants are placed in an upper layer of a column. A single mixture containing nicotinamide, ribose-5-phosphate and ATP is then passed through the column to yield NMN as a final product.

[0072] In another alternative method, resin or beads carrying immobilized PRS mutant enzyme and NAMPT enzyme are mixed into a single column, and a mixture containing nicotinamide, ribose-5-phosphate and ATP is then passed through the column. This latter embodiment will have the advantage of consuming PRPP to further reduce inhibition of PRS enzyme.

[0073] In yet another alternative method, NAMPT is replaced by nicotinate phosphoribosyltransferase and nicotinamide is replaced by nicotinic acid, to yield nicotinic acid mononucleotide (NaMN).

Exemplary Method 2

[0074] The enzyme nicotinamide riboside kinase (NRK) may be purified from cells or produced through recombinant means, and then immobilized on a solid support (e.g. resin, beads). Nicotinamide riboside and ATP are then passed over this solid support to yield nicotinamide mononucleotide (NMN).

Exemplary Method 3

[0075] Glucose, nicotinamide, ATP, NADP.sup.+and an oxidizing agent are passed over a solid support (e.g. resin, beads) which contain the isolated or recombinant enzymes hexokinase, glucose-6-phosphate dehydrogenase, gluconolactonase, 6-phospho gluconate dehydrogenase, ribulose-5-phosphate isomerase, mutant versions of phosphoriboylpyrophosphatase synthetase, and nicotinamide phosphoribosyl transferase. These enzymes may be immobilized to separate solid supports, and placed in layers in the order listed above from top to bottom. Alternatively, all enzymes may be mixed and immobilized to the same solid support. Glucose and nicotinamide will be converted by these enzymes into NMN, which will consume ATP and require the conversion of NADP.sup.+ into NADPH. NADPH will be immediately regenerated back into NADP.sup.+ through the addition of an oxidizing agent.

[0076] The midpoint potential of the NADP.sup.+/NADPH redox pair is 0.324 volts, meaning that NADPH is comparatively easy to oxidize. Thus, preferred oxidizing agents may include any of a number of very mild oxidizing agents known in the art to be capable of oxidizing NADPH into NADP.sup.+.

[0077] The enzymes used in the disclosed systems and methods in all of the above-disclosed exemplary methods may be produced through recombinant means in microbes, such as in yeast, bacteria, baculovirus, or in eukaryotic cells, such as in mammalian cell lines. Methods of producing recombinant enzymes using such host cells are well-known in the art. Alternatively, the enzymes may be produced through in vitro translation methods. A variety of cell-free translation methods are known in the art. A non-limiting example is the use of reticulocyte lysate to facilitate enzyme production.

[0078] Constructs for recombinant expression may be subjected to codon optimization from the parent cDNA to increase protein translation. Again, such techniques are well-known in the art. Although the enzymes described in this disclosure are the human forms, the human form of the enzymes used may be substituted with the orthologous enzymes from other species, depending on the efficiency of their activity.

[0079] Other embodiments and uses will be apparent to those skilled in the art from consideration from the specification and practice of the invention disclosed herein. It is understood that the invention is not confined to the specific reagents, formulations, reaction conditions, etc., herein illustrated and described, but embraces such modified forms thereof as come within the scope of the following claims.

[0080] All references cited herein for any reason, including all journal citations and U.S./foreign patents and patent applications, are specifically and entirely incorporated by reference herein.