WO2020008209A1 - Triphosphorylation reaction - Google Patents

Abstract

The disclosure provides a method of producing a triphosphate compound. The method comprises (a) dissolving imidazole in an aqueous alkaline solution to make an imidazole solution; (b) dissolving phosphoryl halide in an organic solvent to make an organic solution; (c) contacting the imidazole solution and the organic solution to produce a solution comprising phosphorylated imidazole (PIm); (d) removing the organic solvent from the solution comprising PIm; and (e) contacting the PIm with a mutant of nucleoside diphosphate kinase (NDPK) and a diphosphate compound, and thereby producing a triphosphate compound.

Description

Triphosphorylation reaction

The present invention relates to methods of producing triphosphate compounds. In particular, the invention describes how a diphosphate compound may be converted into a triphosphate compound.

Adenosine-5’-triphosphate (ATP) is used as a phosphoryl donor in enzyme-catalysed synthesis, and adenosine-5’-diphosphate (ADP) is produced as a by-product in many such reactions. For example, the preparation of proteins through cell-free in vitro translation systems requires large amounts of ATP. Expensive cofactors, such as NAD(P)H, that would otherwise be used in stoichiometric amounts can also be regenerated through ATP-dependent processes. Oxidoreductases use NAD(P)H as cofactor and economic advantage can thus be gained through NAD(P)H regeneration (Kara, S., Schrittwieser, J.H., Hollmann, F. et al. Appl Microbiol Biotechnol (2014) 98: 1517. https : / / doi.org / io.1007/SOQ253-013-5441-5). Other nucleoside-5’- triphosphates are used as precursors to glyosyl donors, such as UDP-sugars, that are the substrates for glycosyltransferase enzymes. Such glycosyl transferases produce glycosylated products and UDP as a by-product.

ATP is an expensive reactant. Accordingly, the need to constantly feed the above reactions with ATP significantly increases the cost of conducting the reactions. For this reason, the idea of regenerating ATP, by phosphorylating ADP has been explored. Several methods have been developed for re-generating ATP in situ in biochemical and biotechnological processes. These centre on the use of an auxiliary enzyme system and a less expensive phosphoryl donor that is able to (re-)phosphorylate ADP to yield ATP for re-use. Several reviews of the literature are available that summarise work in the area:

a) Enzymatic regeneration of adenosine 5'-triphosphate: Acetyl phosphate,

phosphoenolpyruvate, methoxycarbonyl phosphate, dihydroxyacetone phosphate, 5-phospho-a-d-ribosyl pyrophosphate, uridine-5'- diphosphoglucose. Methods in Enzymology (Vol. 136, pp. 263-280). Elsevier http:/ / doi.org/ 10.1016/50076-6879(87)36027-6

b) Zhao, H., & van der Donk, W. A. (2003). Regeneration of cofactors for use in biocatalysis. Current Opinion in Biotechnology, 14(6), 583-589.

http:/ / doi.org/ 10.1016/j .c0pbi0.2003.09.007 c) Andexer, J. N., & Richter, M. (2015). Emerging Enzymes for ATP Regeneration in Biocatalytic Processes. ChemBioChem, i6{ 3), 380-386.

http:/ / doi.org/ 10.1002/0610.201402550 Whitesides’ work is considered a benchmark of the field. He stated in 1987 (Crans, D. C., Kazlauskas, R. J., Hirschbein, B. L., Wong, C.-H., Abril, O., & Whitesides, G. M. (1987). [25] Enzymatic regeneration of adenosine 5 '-triphosphate: Acetyl phosphate, phosphoenolpyruvate, methoxycarbonyl phosphate, dihydroxyacetone phosphate, 5- phospho-a-d-ribosyl pyrophosphate, uridine-s'-diphosphoglucose. Methods in

Enzymology (Vol. 136, pp. 263-280). Elsevier http://d0i.0rg/10.1016/s0076-

6879(87)36027-6) that three approaches have proven useful for synthetic enzymatic transformations. These approaches are:

1) the use of acetate kinase with acetyl phosphate as phosphoryl donor;

2) the use of pyruvate kinase with phosphoenol pyruvate as phosphoryl donor; and 3) the use of acetate kinase with methoxycarbonyl phosphate as phosphoryl donor.

He compared the effectiveness of the approaches, their advantages and disadvantages. In respect of approach 1, acetyl phosphate has a half-life of only 21 hours, although this material is still used (Yan, B., Ding, Q., Ou, L., & Zou, Z. (2013). Production of glucose- 6-phosphate by glucokinase coupled with an ATP regeneration system. World Journal of Microbiology and Biotechnology, 30(3), 1123-1128. http://d0i.0rg/10.1007/s11274- 013-1534-7)·

In respect of approach 2, while phosphoenol pyruvate has a half-life -40 d, its preparation is complex.

In respect of approach 3, the difficulty of preparation of methoxycarbonyl phosphate is considered to be intermediate between approaches 1 and 3. However, the half-life of the material is only 0.3 h, which is inconveniently short.

More recently, polyphosphate kinases have been employed to regenerate GTP and ATP using polyphosphates as phosphoryl donor. However, they offer limited substrate scope in terms of ability to regenerate other nucleotides (Andexer, J. N., & Richter, M. (2015). Emerging Enzymes for ATP Regeneration in Biocatalytic Processes.

ChemBioChem, 16(3), 380-386. http://d0i.0rg/10.1002/cbic.201402550). The present invention arose from the inventors’ work in attempting to overcome the problems associated with the prior art.

In accordance with a first aspect of the invention, there is provided a method of producing a triphosphate compound, the method comprising contacting

phosphorylated imidazole (PIm) with a mutant of nucleoside diphosphate kinase (NDPK) and a diphosphate compound, and thereby producing a triphosphate compound. Advantageously, the method of the first aspect enables a triphosphate compound to be produced easily and at low cost. The half-life of PIm is known to be 9.6 days (Orth, E. S., Wanderlind, E. H., Medeiros, M., Oliveira, P. S. M., Vaz, B. G., Eberlin, M. N., et al. (2011). Phosphorylimidazole derivatives: potentially biosignaling molecules. The Journal of Organic Chemistry, 76(19), 8003-8008.

http://d0i.0rg/10.1021/j02017394), which compares favourably with acetyl phosphate

(half-life 21 h) and even more favourably with methoxycarbonyl phosphate (half-life 0.3 h).

Prior to contacting the PIm with the mutant of NDPK and the diphosphate compound, the method may comprise preparing the PIm. Preparing the PIm may comprise:

dissolving imidazole in an aqueous alkaline solution to make an imidazole solution;

dissolving a phosphoryl halide in an organic solvent to make an organic solution; and

- contacting the imidazole solution and the organic solution to produce a solution comprising phosphorylated imidazole (PIm).

Preferably, prior to contacting the PIm with the mutant of NDPK and the diphosphate compound, the method comprises removing the organic solvent from the solution comprising PIm. Advantageously, this prevents the organic solvent from denaturing the mutant of NDPK.

The inventors believe that this is an important aspect of the invention. Hence, in accordance with a second aspect, there is provided a method of producing a triphosphate compound, the method comprising: dissolving imidazole in an aqueous alkaline solution to make an imidazole solution;

dissolving phosphoryl halide in an organic solvent to make an organic solution; contacting the imidazole solution and the organic solution to produce a solution comprising phosphorylated imidazole (PIm);

removing the organic solvent from the solution comprising PIm; and contacting the PIm with a mutant of nucleoside diphosphate kinase (NDPK) and a diphosphate compound, and thereby producing a triphosphate compound. It may be appreciated that the phosphoryl halide can have chemical formula POX₃, where each X is a halogen. Each X may independently be fluorine, chlorine, bromine or iodine. In a preferred embodiment, each X is a chlorine. Accordingly, the phosphoryl halide may be phosphoryl chloride. Preferably, the method comprises dissolving the phosphoryl chloride in a dry organic to make the organic solution.

The organic solvent is preferably water miscible. The organic solvent is preferably non- reactive. The organic solvent may comprise an organic nitrile and/ or an ether. The ether may be a cyclic ether. The organic nitrile may be acetonitrile. The ether may be tetrahydrofuran and/or dioxane.

The organic solvent may be removed by evaporation or gel filtration. In a preferred embodiment, the organic solvent is removed by evaporation.

The imidazole may be dissolved in the aqueous solution at a temperature between - 20°C and 150°C, more preferably between o°C and ioo°C, between 5°C and 75°C or between io°C and 50°C, and most preferably between 15°C and 30°C or between 17.5°C and 25°C.

The phosphoryl halide may be dissolved in the organic solvent at a temperature between -20°C and 150°C, more preferably between o°C and ioo°C, between 5°C and 75°C or between io°C and 50°C, and most preferably between 15°C and 30°C or between 17.5°C and 25°C. The imidazole solution and the organic solution may be contacted at a temperature between -20°C and 150°C, more preferably between o°C and ioo°C, between 5°C and 75°C or between io°C and 50°C, and most preferably between 15°C and 30°C or between 17.5°C and 25°C.

The PIm, the mutant of NDPK and the diphosphate compound may be contacted at a temperature between 5°C and 40°C, more preferably between io°C and 37-5°C, between 15°C and 35°C or between 17.5°C and 32.5°C, and most preferably between 20°C and 30°C or between 22.5°C and 27.5°C.

The aqueous alkaline solution preferably comprises a base. The base may be a hydroxide. The hydroxide may be lithium hydroxide, sodium hydroxide or potassium hydroxide. Preferably, the molar ratio of imidazole to phosphoryl chloride is between 1:3 and 3:1 or between 1:2 and 2:1, more preferably is between 1:1.75 and 1.75:1, between 1:1.5 and 1.5:1 or between 1:1.25 and 1.25:1, and most preferably is between 1:1.1 and 1.1:1. In a preferred embodiment, the molar ratio of imidazole to phosphoryl chloride is about 1:1. Preferably, the molar ratio of imidazole to the base is between 1:100 and 5:1 or between 1:50 and 2:1, more preferably is between 1:20 and 1:1, between 1:10 and 1:2 or between 1:8 and 1:4, and most preferably is between 1:7 and 1:5.

Preferably, the molar ratio of phosphoryl chloride to the base is between 1:100 and 5:1 or between 1:50 and 2:1, more preferably is between 1:20 and 1:1, between 1:10 and 1:2 or between 1:8 and 1:4, and most preferably is between 1:7 and 1:5.

Preferably, the imidazole is dissolved in the aqueous alkaline solution to provide an aqueous alkaline solution comprising imidazole, more preferably at a concentration of between 1 and 1000 mM, or between 10 and 500 mM, even more preferably at a concentration of between 25 and 250 mM, or between 50 and 100 mM, and most preferably at a concentration of between 70 and 80 mM.

It maybe appreciated that the phosphorylated imidazole (PIm) in the first and second aspect may be a compound of formula (I):

, or a salt or solvate thereof.

The method may produce a salt of the compound of formula (I).

The method may comprise adjusting the pH of the solution comprising PIm to a predetermined pH. Preferably, the predetermined pH is between 5 and 10, more preferably between 6 and 9, and most preferably between 7 and 8. Without wishing to be bound by theory, the inventors believe at this pH the PIm will exist as a part ionised form, and probably comprise a mixture of the mono- and di-anionic forms.

The mutant of NDPK used in the first and second aspect maybe a mutant of a eukaryotic or prokaryotic NDPK. The mutant of NDPK may be a microbial NDPK, for example a bacterial NDPK, a fungal NDPK or protist NDPK. The bacterial NDPK may be a Gram-positive or a Gram-negative NDPK. The mutant of NDPK maybe from a thermophilic organism or a yeast. In a preferred embodiment, the mutant of NDPK may be from Dictyostelium or Saccharomyces spp. The Saccharomyces spp. is preferably S. cerevisiae.

Preferably, the mutant of NDPK is configured to catalyse the transfer of a phosphate group from a phosphate-donating molecule (e.g. PIm) to a substrate (e.g. a

diphosphate) via a suitable phosphoryl intermediate. It will be appreciated that one embodiment of the consensus sequence of the pocket in NDPK, which phosphorylates a diphosphate group, is provided herein as SEQ ID No:i, as follows:-

HGSDSV

[SEQ ID No: l] However, the inventors have found that due to the imidazole group present in the histidine residue at position 1 in SEQ ID No: 1, the pocket of NDPK is unable to accommodate the phosphorylated imidazole (PIm) in the methods of the invention. As such, preferably the histidine residue at position 1 in SEQ ID No: 1 is modified to an alternative (i.e. a smaller) amino acid residue which does not comprise a ring structure, such as imidazole, to form the mutant NDPK. For example, the wild-type histidine residue may be replaced with an amino acid selected from a group consisting of:

glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, aspartic acid, lysine and asparagine. Preferably, the wild-type histidine residue is replaced with glycine or alanine. Most preferably, the wild-type histidine residue is replaced with glycine.

Accordingly, the mutant of NDPK may comprise or consist of an amino acid sequence substantially as set out in SEQ ID No: 2:

XGSDSV

[SEQ ID No: 2] , wherein X is glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, aspartic acid, lysine or asparagine.

Accordingly, most preferably, the mutant of NDPK is configured to catalyse the transfer of a phosphate group from a phosphate-donating molecule (e.g. PIm) to a substrate (e.g. a diphosphate) via the non-covalent association of the mutant enzyme and PIm, more preferably via a glycine mutation in the wild-type NDPK amino acid sequence (i.e. SEQ ID No:i). Preferably, the mutant of NDPK comprises a glycine mutation in SEQ ID No:i, i.e. the wild-type NDPK amino acid sequence. The wild-type amino acid residue which is mutated to the glycine may be any amino acid, but is preferably a histidine, i.e. a histidine to glycine modification.

Accordingly, the mutant of NDPK may comprise or consist of an amino acid sequence substantially as set out in SEQ ID No: 3: GGSDSV

[SEQ ID No: 3]

In one preferred embodiment, the mutant of NDPK is a mutant of Dictyostelium NDPK. This may also be known as NDPK H122G. In one embodiment, the amino acid sequence of the mutant of Dictyostelium NDPK is provided herein as SEQ ID No: 4, as follows :- MSTNKVNKERTFLAVKPDGVARGLVGEI IARYEKKGFVLVGLKQLVPTKDLAESHYAEHKERPFFGGLVS FITSGPVVAMVFEGKGVVASARLMIGVTNPLASAPGSIRGDFGVDVGRNI IGGSDSVESANREIALWFKP EELLTEVKPNPNLYE

[SEQ ID No: 4]

Preferably, therefore, the mutant of NDPK comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 4, or a fragment or variant thereof.

Most preferably, the mutant of NDPK used in the first and second aspect, is NDPK H122G. It may be appreciated that, in this mutation, a glycine is provided at position

122 instead of a histidine (as in the wild-type Dictyostelium enzyme). In some embodiments, the mutant of NDPK may further comprise a peptide tag to facilitate enzyme purification, for example MAHHHHHHSSGLEVLFQGP (SEQ ID No. 5), which maybe disposed as an N-terminal tag.

In another preferred embodiment, the mutant of NDPK is a mutant of S. cerevisiae NDPK. This may also be known as NDPK H119G. In one embodiment, the amino acid sequence of the mutant of S. cerevisiae NDPK is provided herein as SEQ ID No: 6, as follows :-

MSSQTERTFIAVKPDGVQRGLVSQILSRFEKKGYKLVAIKLVKADDKLLEQHYAEHVGKPFFPKMVSFMK SGPILATVWEGKDVVRQGRTILGATNPLGSAPGTIRGDFGIDLGRNVCGGSDSVDSAEREINLWFKKEEL VDWESNQAKWIYE

[SEQ ID No: 6]

Preferably, therefore, the mutant of NDPK comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 6, or a fragment or variant thereof. Preferably, the mutant of NDPK used in the first and second aspect, is NDPK H119G. It may be appreciated that, in this mutation, a glycine is provided at position 119 instead of a histidine (as in the wild-type S. cerevisiae enzyme).

In a preferred embodiment, the diphosphate compound in the first and second aspect is a nucleoside diphosphate, or a salt or solvate thereof. Accordingly, the methods of the first or second aspects may comprise producing a nucleoside triphosphate, or a salt or solvate thereof. The nucleoside diphosphate maybe a compound of formula (II):

, wherein Base is a nucleobase;

X is S, CH₂, or O;

R¹ is hydrogen, a C -C₆ alkyl, a halogen, azido or cyano;

R² is hydrogen, a halogen, azido, cyano, NR⁷R⁸ or OR⁷;

R3 is hydrogen, a halogen, azido, cyano, NR⁷R⁸ or OR⁷;

R4 is hydrogen, a C -C₆ alkyl, a halogen, azido or cyano;

R⁵ is hydrogen, azido or a halogen;

R⁶ is hydrogen or a C -C₆ alkyl; and

the or each R⁷ and R⁸ are independently hydrogen or a C -C₆ alkyl optionally substituted with a halogen, azido or cyano;

or a salt or solvate thereof.

Accordingly, the nucleoside triphosphate in the first and second aspect may be a compound of formula (III):

, or a salt of solvate thereof.

The Base may be purine, pyrimidine or a derivative thereof.

The purine or pyrimidine derivative may be adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(0)(alkyl, aryl, alkylaryl, or arylalkyl)), N⁶-benzylpurine, N⁶- halopurine, N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkyl purine, N⁶-alkylaminopurine, N⁶-thioalkyl purine, N²-alkylpurines, N²-alkyl-6- thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil, C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵- halopyrimidines, C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵- hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine, C⁵-iodopyrimidine, C⁶- iodo-pyrimidine, C⁵-Br-vinyl pyrimidine, C⁶-Br-vinyl pyrimidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine, N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and

pyrazolopyrimidinyl. Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 7-deazaguanine, 7-deazaadenine, 2,6-diaminopurine, and 6- chloropurine. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t- butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.

The purine derivative maybe adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine or 7-methylguanine. The pyrimidine derivate may be cytosine, thymine or uracil. Alternatively, or additionally, the nucleobase may be labelled with a fluorophore and/ or dye. Accordingly, the base may be substituted with a linker and a fluorophore and/ or dye. Examples of the labelled bases are given in a review article (Chen, F., Dong, M.,

Ge, M., Zhu, L., Ren, L., Liu, G., & Mu, R. (2013). The History and Advances of

Reversible Terminators Used in New Generations of Sequencing Technology.

Genomics, Proteomics & Bioinformatics, 11(1), 34-40.

http:/ / doi.org/ 10.1016/j .gpb.2013.01.003).

Preferably, X is O. Preferably, R¹ is hydrogen.

Preferably, R² is a hydroxyl or a halogen. The halogen may be fluorine, chlorine or bromine, more preferably fluorine or chlorine, and most preferably fluorine.

Preferably, R² is a hydroxyl. Preferably, R³ is a hydroxyl or a halogen. The halogen may be fluorine, chlorine or brome, more preferably fluorine or chlorine, and most preferably fluorine. Preferably, R³ is a hydroxyl. Preferably, R⁴ is hydrogen.

Preferably, R⁵ is hydrogen.

Preferably, R⁶ is hydrogen.

Accordingly, the nucleoside diphosphate is preferably a compound of formula (Ila):

, or a salt or solvate thereof.

Furthermore, the nucleoside triphosphate is preferably a compound of formula (Ilia):

, or a salt or solvate thereof.

Preferably, Base is purine or a derivative thereof.

The method of producing a triphosphate compound of the first or second aspect maybe conducted in situ to a parallel reaction converting the triphosphate compound to a diphosphate compound. The parallel reaction maybe an enzyme-catalysed synthesis reaction. Advantageously, recycling the diphosphate compound to generate more of the triphosphate compound allows a smaller quantity to be used, reducing the cost of the parallel reaction. The parallel reaction maybe an enzyme-catalysed synthesis reaction.

For instance, glycosylation processes frequently use nucleoside-5’-triphosphates (NTPs) sugars to form nucleoside-5’-diphosphate-sugar (NDP-sugar).

Glycosyltransferase enzymes then catalyse the transfer of sugar from NDP-sugar to a substrate. This results in the formation of a nucleoside-5’-diphosphate (NDP) by- product that can be recycled to NTP for re-use in the formation of NDP-sugars using the methods of the first and second aspect.

In one embodiment, Base is cytosine. Accordingly, the nucleoside diphosphate may be cytidine-5’-diphosphate (CDP), or a salt or solvate thereof. Accordingly, the method maybe a method of producing cytidine-5’-triphosphate (CTP), or a salt or solvate thereof.

In an alternative embodiment, Base is thymine. Accordingly, the nucleoside diphosphate may be 5-methyluridine-5’-diphosphate (rmUDP), or a salt or solvate thereof. Accordingly, the method maybe a method of producing 5-methyluridine-5’- triphosphate (rmUTP), or a salt or solvate thereof.

In a further alternative embodiment, Base is uracil. Accordingly, the nucleoside diphosphate may be uridine-5’-diphosphate (UDP), or a salt or solvate thereof.

Accordingly, the method maybe a method of producing uridine-5’-triphosphate (UTP), or a salt or solvate thereof.

In a further alternative embodiment, Base is guanine. Accordingly, the nucleoside diphosphate may be guanosine-5’-diphosphate (GDP), or a salt or solvate thereof. Accordingly, the method maybe a method of producing guanosine-5’-triphosphate (GTP), or a salt or solvate thereof.

In further alternative embodiment, Base is adenine. Accordingly, the nucleoside diphosphate may be adenosine-5’-diphosphate (ADP), or a salt or solvate thereof. Accordingly, the method maybe a method of producing adenosine-5’-triphosphate (ATP), or a salt or solvate thereof. In accordance with a third aspect, there is provided a method of producing adenosine- 5’-triphosphate (ATP), the method comprising contacting phosphorylated imidazole (PIm) with a mutant of nucleoside diphosphate kinase (NDPK) and adenosine-5’- diphosphate (ADP), and thereby producing ATP.

The PIm maybe produced as described in relation to the first and second aspects.

The PIm, mutant of NDPK and ADP may be contacted at a temperature between 5°C and 40°C, more preferably between io°C and 37-5°C, between 15°C and 35°C or between 17.5°C and 32.5°C, and most preferably between 20°C and 30°C or between 22.5°C and 27.5°C.

The method of producing ATP may be conducted in situ to a parallel reaction converting ATP to ADP. The parallel reaction may be an enzyme-catalysed synthesis reaction. Advantageously, recycling the ADP to produce more ATP allows a smaller quantity to be used, reducing the cost of the parallel reaction. The parallel reaction may be an enzyme-catalysed synthesis reaction.

For instance, enzymatic carbon-carbon bond forming and cleavage processes often employ aldolase enzymes that require sugar mono-phosphates as substrates. Sugar mono-phosphates are formed through the actions of sugar kinases that use ATP as a source of a phosphoryl group, and yield ADP as a by-product. The ADP can be recycled to ATP for re-use in the formation of sugar mono-phosphates using the method of the third aspect.

Preferably, the method of the first, second or third aspect comprises disposing PIm, the mutant of NDPK and the diphosphate compound in a further solution, and thereby contacting the PIm, the mutant of NDPK and the diphosphate compound. It may be appreciated that in embodiments where the diphosphate compound is ADP the further solution may also comprise the reagents required for the parallel reaction. Accordingly, the further solution may comprise ATP. The further reaction may also comprise an enzyme configured to catalyse the further reaction. It may be appreciated that the PIm may not be prepared in the further solution.

Preparing the PIm in a separate solution allows the organic solvent to be removed prior to the PIm being disposed in the further solution. Advantageously, this prevents NDPK, and optionally the enzyme configured to catalyse the further reaction, from being denatured. The further solution may be an aqueous solution.

The PIm and the mutant of NDPK maybe contacted at a temperature between 5°C and 40°C, more preferably between io°C and 37-5°C, between 15°C and 35°C or between 17.5°C and 32.5°C, and most preferably between 20°C and 30°C or between 22.5°C and 27.5°C.

Preferably, the concentration of the PIm in the further solution is at least i mM, at least to pM or at least 100 pM, more preferably at least l mM, at least 10 mM or at least 50 mM, and most preferably at least 0.1 M. Preferably, the concentration of the PIm in the further solution is between 1 pM and 10 M, between 10 pM and 7.5 M or between 100 pM and 5 M, more preferably a concentration of between 1 mM and 3 M, between 10 mM and 2 M or between 100 mM and 1 M, and most preferably a concentration of between 0.1 and 0.6 M. Preferably, the concentration of the diphosphate compound in the further solution is at least 0.01 pM, at least 0.1 pM, at least 1 pM or at least 5 pM, more preferably at least 10 pM, at least 25 pM or at least 50 pM, and most preferably at least 100 pM. Preferably, the concentration of the diphosphate compound in the further solution is between 0.01 pM and 10 mM, between 0.1 pM and 7 mM, between 1 pM and 4 mM or between 5 pM and 2 mM, more preferably a concentration of between 10 pM and 1 mM, between 25 pM and 750 pM or between 50 pM and 500 pM, and most preferably a concentration of between 100 and 300 pM.

Preferably, the molar ratio of PIm to the diphosphate compound in the further solution is between 1:1 and 1,000,000:1 or between 10:1 and 100,000:1, more preferably is between 50:1 and 10,000:1 or between 100:1 and 5,000:1, and most preferably is between 400:1 and 2500:1.

Preferably, the concentration of the mutant of NDPK in the further solution is at least 0.01 ng/ml or at least 0.1 ng/ml, more preferably at least 1 ng/ml or at least 5 ng/ml, and most preferably is at least 10 ng/ml. Preferably, the concentration of the mutant of NDPK in the further solution between o.oi and 5000 ng/ml or between 0.1 and 1000 ng/ ml, more preferably between 1 and too ng/ ml or between 5 and 50 ng/ ml, and most preferably is between 10 and 30 ng/ml. The method may comprise adjusting the pH of the further solution to a predetermined pH. Preferably, the predetermined pH is between 5 and 10, more preferably between 6 and 9, and most preferably between 7 and 8. Preferably, the method comprises maintaining the further solution at the predetermined pH. Preferably, the method comprises disposing the PIm in the solution prior to disposing the NDPK therein. Preferably, the pH of the further solution is adjusted prior to disposing the mutant of NDPK therein. The pH of the further solution may be adjusted after disposing the PIm therein. Advantageously, adjusting or maintaining the pH of the solution ensures the solution is at a pH where the mutant of NDPK is active.

Preferably, the pH of the solution is adjusted and/or maintained by contacting the solution with a buffer. The buffer is preferably configured to maintain the solution at the predetermined pH. The buffer may comprise 4-(2-hydroxyethyl)-i- piperazineethanesulfonic acid (HEPES) or tricine.

Alternatively, or additionally, the pH of the solution is adjusted by contacting the solution with an acid. The acid may comprise hydrochloric acid, sulphuric acid and/or acetic acid. It will be appreciated that the invention extends to any method as defined above which use a nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms“substantially the amino acid/nucleotide/peptide sequence”,“variant” and“fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/ nucleotide/ peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID Nos: 1-6 and so on.

Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino

acid/polynucleoti de/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.

The skilled technician will appreciate howto calculate the percentage identity between two amino acid/polynucleoti de/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleoti de/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et ah, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et ah, 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW maybe as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -l, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino

acid/ polynucleotide/ polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the

ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N/T)^*ioo. Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C.

Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or too amino acids from the sequences shown in, for example, SEQ ID Nos:i to 6. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.

Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a

conservative change. For example small non-polar, hydrophobic amino acids include gfycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

All features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which: -

Figure 1 shows the reaction scheme and products for the reaction of imidazole (Im) in a sodium hydroxide (NaOH) solution when contacted with phosphoryl chloride (POCl₃) in dry acetonitrile (MeCN);

Figure 2 is a graph showing how the percentage of phosphorylated imidazole (PIm) produced varies as the concentration of Im varies in the initial reaction shown in Figure

1;

Figure 3 shows HPLC chromatograms obtained for a reaction of PIm and guanosine diphosphate (GDP);

Figure 4 shows HPLC chromatograms obtained for a further reaction of PIm and guanosine diphosphate (GDP);

Figure 5 is a graph showing the percentage of GDP converted to guanosine

triphosphate (GDP) against time;

Figure 6 is a schematic diagram illustrating a phosphorylation reaction;

Figure 7 shows HPLC chromatograms obtained for a reaction of PIm and adenosine- 5’-diphosphate (ADP); and

Figure 8 is a graph showing the relative proportion of ATP and ADP after initiation of a regeneration reaction. Example l: Synthesis of phosphorylated imidazole fPIm) using a 75 mM concentration of imidazole f Im)

Imidazole (0.51 g, 7.5 mmol, 1 equivalent) was dissolved in sodium hydroxide (100 ml, 450 mM, 45 mmol, 6 equivalents) under an inert atmosphere. Dry acetonitrile (10 ml) was added to phosphoryl chloride (699 pL, 7.5 mmol, 1 equivalent) in a dropping funnel, under an inert atmosphere. The phosphoryl chloride solution was added dropwise to the stirred imidazole solution of imidazole over 10 minutes and stirring was maintained for 1 h after the final addition. A small aliquot of the material was then analysed by 31-P NMR spectroscopy in order to assess the product distribution in the crude reaction solution.

Example 2: Optimisation of reagent conditions

The procedure described in example 1 was repeated using a range of concentrations of imidazole with the aim to improve conversion to phosphorylated imidazole (PIm). The same 1:6:1 stoichiometry of Im:NaOH:POCl₃ was maintained across these experiments. Experiments were conducted using concentrations of 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM and 500 mM. Crude reaction mixtures were then analysed by 31-P NMR spectroscopy. Crude reaction mixtures contained a mixture of species as follows: d_r (D₂0) -4.5 (iP,s, Pi), 5.0 (iP,s, PIm), 8.2 (lP, s, Bis-Im-P). The signals for these species were integrated and the ratios of integrals were used to determine the ratios of P-containing products, and the results are shown in table 1 and figure 2.

Table 1: The % of reactants formed after one hour of stirring

While both the 75 mM and the 250 mM reactions gave similarly high levels of conversion to PIm of 55%, the 250 mM reaction was extremely vigorous to the extent that a pressure release was necessary to prevent the reaction vessel from causing the clipped on dropping funnel to‘pop’ off. As such the 75 mM reaction was chosen as the most convenient route to access PIm.

Example 2: Isolation of PIm

After formation of the crude PIm as described above in examples 1 and 2, the reaction mixture was then subjected to rotary evaporation (-45 minutes) to remove the acetonitrile and some of the water - around 90 ml of liquid was taken off via rotary evaporation at this stage. Rotary evaporation results in the formation of a white precipitate. The precipitate and liquor were then separated via centrifugation (4000 rpm, 10 minutes). The precipitate was then recrystallised twice from hot deionised water (3 ml), after which the precipitate was dried on the high vacuum line. The resulting solid was extracted with ethyl acetate (50 ml) by vigorous overnight stirring to remove unreacted imidazole (detected by l-H NMR spectroscopy). The solid was collected, dried on the high vacuum line and stored in the freezer.

The crude reaction mixtures of the 75 mM, 150 mM and 200 mM PIm synthesis experiments were analysed after centrifugation via 31P NMR spectroscopy to determine the composition of the precipitate and the supernatant. In all three cases, the precipitate was found to have PIm as the main component, with the 75 mM iteration being made up of 76% PIm, as shown in table 2.

Table 2: The % of components found in the supernatant and precipitate after rotary evaporation

Example 4: Reaction of PIm and guanosine diphosphate (GDP)

To 1 ml reaction buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 10 mM MgCl₂) was added 5 mΐ too mM guanosine diphosphate (GDP) and 75 mg PIm. The pH was adjusted to 7-8 using hydrochloric acid. NDPK H122G enzyme (34 mΐ 0.6 mg/ml) was added to the mixture, then too mΐ samples were removed at 1 minute, 10 minutes, 30 minutes and 60 minutes for HPLC analysis. Before HPLC analysis, enzyme was removed using an Amicon 10 kDa MWCO spin column at 13.5 rpm. A reaction with no enzyme was also performed in a similar manner. The reactions were conducted at room temperature.

Using the HPLC chromatograms, shown in Figure 3, the inventors were able to confirm that guanosine triphosphate GTP was formed when enzyme was present, but not in the absence of enzyme or in the absence of PIm. The overall reaction is shown

schematically in Figure 6 where NDP is GDP.

Example : Further reaction of PIm and GDP

The inventors repeated the experiment described in example 4, but with less PIm (15 mg), and the experiment was allowed to run for 47 hours. All other conditions were kept the same.

The HPLC chromatograms up until 23 hours are shown in Figure 4. The chromatogram obtained after 23 hours shows a particularly diminished signal at 9 minutes, which corresponds to GDP, and a particularly enhanced signal at 21 minutes, which corresponds to GTP. The HPLC areas were integrated to allow the amount of GDP which had been converted to GTP to be calculated, and are shown in table 3 and Figure

4·

Table 2: Amount of GDP which was converted to GTP as the reaction progressed

Example 6 - Reaction of PIm and adenosine-A-diphosphate fADP)

In an analogous fashion to reaction of PIm and guanosine-5’-diphosphate in Example 5, the formation of ATP from ATP was demonstrated by replacing GDP with ADP. Using the HPLC chromatograms, shown in Figure 7, the inventors were able to confirm that adenosine triphosphate ATP was formed when enzyme was present. The overall reaction is shown schematically in Figure 6 where NDP is ADP. Illustrative HPLC chromatograms at an early time point of ~o.i hours and a later timepoint of 24 hours are shown in Figure 7. The chromatogram obtained after 24 hours shows a particularly diminished signal at 7 minutes, which corresponds to ADP, and a particularly enhanced signal at 5.7 minutes, which corresponds to ATP. The HPLC areas of a series of experiments were integrated to allow the amount of ADP which had been converted to ATP to be calculated, and are shown in table 4 and Figure 8.

Table d: Relative proportion of ATP and ADP after initiation of the regeneration reaction

Example 7 - In situ generation of ATP from ADP and consumption of the ATP bv another enzyme

In order to demonstrate the ability on the inventors’ system to produce ATP

advantageously for use by another enzyme, ADP was converted to ATP which was consumed by a luciferase enzyme. Luciferase employs ATP together with D-luminol and atmospheric oxygen to generate a luminescence output. A Molecular Probes/Invitrogen ATP detection kit (A22066) was calibrated by adding 10 pL of 5 pM, 0.5 pM, 50 nM, 5 nM and 0.5 nM ATP to too pL of the assembled assay system. Proportional

luminescence responses were seen for all but the lowest concentration, which appears to give a signal below the background level.

A series of experiments was performed using NDPK H122G from a stock solution of~o.2 mg/mL. Five experiments were run across the wells of a black-bodied microtiter plate at 28 °C. In addition to too pL of assembled assay system, wells contained ADP, crude PIm and/or enzyme stock, as shown in table 5. Table 5: Reagents added to the assay system for experiments Cl to CA

Luminescence was measured every minute for 10 minutes and the results are shown in table 6. Table 6: Luminescence of experiments Cl to C

The positive control experiment (C2) gave the largest signal, and this differentiation increased with time as more ATP was formed in situ. The background response appeared high in negative controls (C3 and C4), which was attributed to the likely presence of low levels of ATP contaminating ADP owing to the method used to extract and isolate commercial nucleotides from whole organisms.

Example 8 - Reaction of PIm and ADP, using a lower concentration of ADP

In order to reduce the background response observed in the negative control (C3 and C4) in example 6, a 100-fold lower ADP concentration was explored in experiments Di to D5. The experiments were conducted in the same way as described in example 7. In addition to too pL of assembled assay system, wells contained ADP, crude PIm and/or enzyme stock, as shown in table 7. Table 7: Reagents added to the assay system for experiments Pi to D

Luminescence was measured every minute for 10 minutes and the results are shown in table 8. Table 8: Luminescence of experiments Pi to D

The background response was significantly lower for the negative controls (D3 and D4). However, the positive control (D2) exhibited a response which was only ~2-4-fold above the negative control level.

Example Q - Reaction of PIm and ADP, using a higher concentration of enzyme

In order to increase the foreground enzymatic process, the inventors used an 8-fold higher concentration of enzyme (where applicable). The experiments were conducted in the same way as described in example 7. In addition to too pL of assembled assay system, wells contained ADP, crude PIm and/or enzyme stock, as shown in table 9. Table Q: Reagents added to the assay system for experiments El to Es

Luminescence was measured every minute for 10 minutes and the results are shown in table to.

Table to: Luminescence of experiments El to

The foreground response was larger and, critically, the luminescence signal for positive control (E2) increased with time, indicating the build-up of ATP and thus a larger luminescence signal at later time points. Conversely, the signal from control E3, although above background because of ATP contamination, remained constant over the short timescale of the reaction.

Example 10 - Reaction of PIm and ADP. using a higher concentration of PIm

The rate of ATP generation (and thus luminescence signal) was also expected to be dependent upon PIm concentration, thus a further experiment was performed with increased PIm concentration and partially elevated enzyme concentration.

The experiments were conducted in the same way as described in example 7. In addition to 100 pL of assembled assay system, wells contained ADP, crude PIm and/or enzyme stock, as shown in table 11. Table 11: Reagents added to the assay system for experiments Hi to Hf;

Luminescence was measured every minute for 10 minutes and the results are shown in table 12.

Table 12: Luminescence of experiments Hi to Hf;

A marked increase in signal was observed through the combination of raising the concentration of both the PIm and enzyme.

Example 11 - Reaction of PIm and ADP over longer time intervals

ATP arising from contamination was expected to be consumed at longer timescales by luciferase in ATP detection kit. Accordingly, this consumption was expected to lead to a reduced luminescence signals from control wells. On the other hand, the experiments containing the inventors’ system (e.g. E2 and H2) were expected to continue to produce ATP thus leading to a maintained or indeed increased luminescence signal at longer incubation times.

Thus the luminescence signals for experiments E and H were re-assessed after extended incubation to confirm this hypothesis. Experiments E1-5 were remeasured approximately 1.25 h after initiation of the reaction. At this time, luminescence was again measured every minute for 10 minutes and the results are shown in table 13. Table 12: Luminescence of experiments El to E^ remeasured approximately 1.25 hours after initiations of the reaction

The remeasurement of luminescence signals after 1.25 h showed that controls E3 and E4 had now fallen to background level because any ATP contaminant had been fully consumed. The luminescence signal from E2, however, was still in the order of the value measured -1.25 h earlier, indicating that a steady state concentration of ATP had been reached and that the luminescence signal was due wholly to ATP produced in situ by the inventors’ system. Experiments H1-5 were remeasured approximately 0.25 h after initiation of the reaction. At this time, luminescence was again measured every minute for 10 minutes and the results are shown in table 14.

Table 14: Luminescence of experiments Hi to H ; remeasured approximately 0.25 hours after initiations of the reaction

The control experiment signals H3 and H4 had dropped significantly owing to the consumption of ATP contaminants. Meanwhile, the signal for H2 had grown substantially owing to the increased build-up of ATP from the inventors’ system.

Example 12 - Further reaction of PIm and ADP over longer time intervals

In order to further confirm the continued generation of ATP by the inventors’ system, and also its longevity, the microtiter plate containing all the above experiments was stored overnight at 4 °C then allowed to equilibrate to room temperature. The luminescence signals were then re-read at a single time point, and the results are given in table 15.

Table 15: Luminescence of all experiments remeasured after the overnight storage of the microtiter plate containing at 4 °C

The C experiments show that controls C3 and C4 diminished in intensity significantly, whereas the signal C2 shows only ~2-fold reduction in luminescence signal. The D experiments show only a small signal above background for D2. However, the initial signal was small. The signal for D2 is also ~2-fold lower than observed in the initial reading. Controls D3 and D4 have diminished to background. The E experiments show ~75% of the original luminescence signal for E2, and controls remain at background levels. Finally, the H experiments showed a very large luminensce signal from the inventors’ system of ~200-fold above background and ~33% of the peak value that was measured.

Conclusion

The inventors have developed a new method of phosphorylating a diphosphate compound. The inventors have shown that the method can be used to regenerate ATP in situ to a parallel reaction which is consuming ATP. Accordingly, the reaction can be used to reduce the overall cost of a parallel biosynthesis reaction which consumes ATP.

Claims

Claims

1. A method of producing a triphosphate compound, the method comprising: dissolving imidazole in an aqueous alkaline solution to make an imidazole solution;

dissolving phosphoryl halide in an organic solvent to make an organic solution; contacting the imidazole solution and the organic solution to produce a solution comprising phosphorylated imidazole (PIm);

removing the organic solvent from the solution comprising PIm; and

- contacting the PIm with a mutant of nucleoside diphosphate kinase (NDPK) and a diphosphate compound, and thereby producing a triphosphate compound.

2. A method according to claim l, wherein the phosphoryl halide is phosphoryl chloride.

3. A method according to claim l or claim 2, wherein the organic solvent is water miscible.

4. A method according to claim 3, wherein the organic solvent comprises an organic nitrile and/ or an ether.

5. A method according to claim 4, wherein the organic solvent comprises acetonitrile. 6. A method according to any preceding claim, wherein the aqueous alkaline solution comprises a base.

7. A method according to claim 6, wherein the base comprises a hydroxide, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide.

8. A method according to either claim 6 or claim 7, wherein the molar ratio of imidazole to the base is between 1:100 and 5:1.

9. A method according to any preceding claim, wherein the molar ratio of imidazole to phosphoryl chloride is between 1:3 and 3:1.

10. A method according to any preceding claim, wherein the diphosphate compound is a nucleoside diphosphate, or a salt or solvate thereof. li. A method according to claim 10, the nucleoside diphosphate is 5-methyluridine- 5’-diphosphate (msUDP), uridine-5’-diphosphate (UDP), guanosine-5’-diphosphate (GDP), adenosine-5’-diphosphate (ADP), or a salt or solvate thereof.

12. A method of producing adenosine-5’-triphosphate (ATP), the method comprising contacting phosphorylated imidazole (PIm) with a mutant of nucleoside diphosphate kinase (NDPK) and adenosine-5’-diphosphate (ADP), and thereby producing ATP.

13. A method according to claim 12, wherein the method of producing ATP is conducted in situ to a parallel reaction converting ATP to ADP.

14. A method according to any preceding claim, wherein the mutant of NDPK is a mutant of a eukaryotic or prokaryotic NDPK. 15. A method according to any preceding claim, wherein the mutant of NDPK comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 2, wherein X is glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, aspartic acid, lysine or asparagine. 16. A method according to claim 15, wherein the mutant of NDPK comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 3.

17. A method according to claim 16, wherein the mutant of NDPK is a mutant of Dictyostelium NDPK and comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 4.

18. A method according to any preceding claim, wherein the method comprises disposing PIm, the mutant of NDPK and the diphosphate compound in a further solution, and thereby contacting the PIm, the mutant of NDPK and the diphosphate compound.

19. A method according to claim 18, wherein the method comprises adjusting the pH of the further solution to a predetermined pH between 5 and 10, between 6 and 9, or between 7 and 8. 20. A method according to claim 19, wherein the method comprises maintaining the further solution at the predetermined pH.