JP6261166B2 - Method for imparting polyphosphate utilization to ATP-dependent kinase - Google Patents

Description

  The present invention relates to a method for imparting polyphosphate utilization to an ATP-dependent kinase, and a kinase mutant having polyphosphate utilization.

  Conventionally, when producing useful phosphorylated compounds, ATP has been used as a phosphorylating enzyme (ATP-dependent kinase) that uses only ATP and a phosphate donor (energy carrier). However, ATP is expensive, and reaction by-products such as ADP are produced. In addition, there are cases where it is difficult to separate the product from unreacted ATP and by-product ADP. Not ideal. On the other hand, polyphosphoric acid is a polymer compound in which phosphoric acid is bound by a phosphate ester bond, and the energy per ester bond is assumed to be equal to that of ATP. Polyphosphate is expected from an industrial point of view as an ideal phosphate donor for the enzymatic production of phosphorylated compounds. This is because polyphosphoric acid is cheaper than ATP, and a highly pure end product can be obtained easily and economically.

  Non-Patent Document 1 reports that large-scale industrial production of NADP was successful using polyphosphate / ATP-NAD kinase. Polyphosphate / ATP-NAD kinase is an enzyme that phosphorylates NAD using polyphosphate and ATP. However, most of the existing phosphorylating enzymes (kinases) are ATP-dependent kinases, and the types of enzymes that can use polyphosphate are limited. In addition to polyphosphate-dependent NAD kinase and the same glucokinase (polyphosphate is used to phosphorylate NAD or glucose to produce NADP or glucose-6-phosphate, respectively), about 10 types of enzymes are known. (Non-patent document 2). Among these, a NADP production method using polyphosphate-dependent NAD kinase and polyphosphate has been reported (Patent Document 1). This NADP production method is an excellent method for producing higher-purity NADP at a lower cost than the conventional method using ATP.

  If a methodology for imparting polyphosphate utilization to other ATP-dependent kinases is established, it is expected that various useful phosphorylated compounds can be produced at a lower cost and with a higher purity. However, there is no known methodological method for imparting polyphosphoric acid utilization ability, and it was unclear whether it was actually possible.

Patent No. 4088251

Kawai, S .; et al. , J .; Biosci. Bioeng. 92, 447-452 (2001) Rao, N .; N. , Gomez-Garcia, M .; R. & Kornberg, A.M. Annu. Rev. Biochem. 78,605-647 (2009)

  An object of the present invention is to provide a means for imparting polyphosphate utilization to an ATP-dependent kinase.

  The inventors have shown that the GGDG motif is highly conserved in the primary structure of phosphorylases including NAD kinase, 6-phosphofructokinase and diacylglycerol kinase, the Asn adjacent to the GGDG motif in ATP-dependent kinases. The present inventors have found that polyphosphate utilization ability can be imparted to ATP-dependent kinases by replacing the residue with a Thr residue.

That is, the present invention includes the following.
(1) A method for imparting polyphosphate utilization to an ATP-dependent kinase,
In the ATP-dependent kinase consisting of an amino acid sequence containing a GGDGN motif, the method comprises replacing the Asn residue of the GGDGN motif with a Thr residue.
(2) The method according to (1), wherein the ATP-dependent kinase is an ATP-dependent NAD kinase.
(3) The method according to (1) or (2), wherein the ATP-dependent kinase is derived from γ proteobacteria.
(4) The method according to any one of (1) to (3), wherein the Asn residue of the GGDGN motif is an Asn residue at a position corresponding to position 75 when SEQ ID NO: 1 is used as a reference sequence.
(5) A kinase mutant having an ability to use polyphosphate, comprising an amino acid sequence in which an Asn residue of a GGDGN motif is substituted with a Thr residue in an amino acid sequence containing a GGDGN motif encoding an ATP-dependent kinase.
(6) The kinase mutant according to (5), wherein the ATP-dependent kinase is an ATP-dependent NAD kinase, and the kinase mutant is a NAD kinase mutant.
(7) The kinase mutant according to (5) or (6), wherein the ATP-dependent kinase is derived from γ-proteobacteria.
(8) The kinase variant according to any one of (5) to (7), wherein the Asn residue of the GGDGN motif is an Asn residue at a position corresponding to position 75 when SEQ ID NO: 1 is used as a reference sequence .
(9) Kinase variant comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 8 or 10, and (b) an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 8 or 10, and having a GGDGT motif, and polyphosphate Protein having activity of phosphorylating NAD using (c) In the amino acid sequence of SEQ ID NO: 8 or 10, one or several amino acids are deleted, substituted, inserted or added, but the GGDGT motif is retained A protein comprising an amino acid sequence and having an activity of phosphorylating NAD using polyphosphate.
(10) A DNA encoding the kinase variant according to any one of (5) to (9).
(11) A transformant comprising the DNA according to (10).
(12) A method for producing NADP, comprising polyphosphoric acid or a salt thereof and NAD as a substrate, and performing a phosphorylation reaction using the kinase mutant according to any one of (5) to (9), Said method.

  According to the present invention, polyphosphate utilization ability can be imparted to an ATP-dependent kinase, and a kinase mutant having polyphosphate utilization ability can be obtained. Thereby, various useful phosphorylated compounds can be produced at a lower cost and with a higher purity.

1 shows alignment of primary structures of ATP-NADK, polyphosphate / ATP-NADK and γ-proteobacterial NADK homolog. The results of assaying ATP-dependent or polyphosphate-dependent NADK activity of wild-type or mutant NADK are shown. The up and down arrows indicate the increase and decrease of NADK activity with each substitution, respectively. It is a bar graph which shows ratio (%) of polyphosphate dependent NADK activity with respect to ATP dependent NADK activity of an ecoNADK variant. The results of incubation of the NADK reaction mixture and analysis by TLC are shown.

  In one embodiment, the present invention relates to a method for conferring polyphosphate utilization to an ATP-dependent kinase, and the method is an ATP-dependent kinase consisting of an amino acid sequence containing a GGDGN motif (hereinafter referred to as an ATP-dependent kinase containing a GGDGN motif). The Asn residue of the GGDGN motif is replaced with a Thr residue.

  In the present invention, a kinase refers to an enzyme having an activity of transferring (phosphorylating) a phosphate group from a phosphate donor having a high energy phosphate bond to a substrate. The ATP-dependent kinase to which polyphosphate utilization ability is imparted is not particularly limited as long as it is an enzyme having an amino acid sequence containing a GGDGN motif and having an activity of transferring a phosphate group from ATP to a substrate.

  ATP dependence in ATP-dependent kinase (ATP-NADK) means that only ATP is used as a phosphate donor in the phosphorylation reaction. ATP-dependent kinase cannot substantially use polyphosphate as a phosphate supplier. For example, the reaction rate with respect to polyphosphate is 15% or less, particularly 12% or less of the reaction rate with respect to ATP.

  Examples of the ATP-dependent kinase containing the GGDGN motif include ATP-dependent NAD kinase. NAD kinase is an enzyme having the activity of transferring the phosphate group of a phosphate donor to the 2'-position of the ribose of adenylate of NAD to produce NADP. That is, ATP-dependent NAD kinase can catalyze the following phosphorylation reaction that produces NADP from NAD using ATP as a phosphate donor.

NAD is one of the coenzymes involved in oxidoreductase, and the reduced form is NADH. It is also called diphosphopyridine nucleotide (DPN, DPNH), coenzyme, coenzyme I (CoI), etc., and is the coenzyme present in the body in the largest amount. NAD has a structure in which nicotinamide mononucleotide (NMN) and adenylic acid are linked by a phosphodiester bond. Since the nitrogen atom of the pyridine ring in oxidized NAD exists as a pyridium ion, this is expressed as NAD ⁺ . The biosynthesis of NAD ⁺ is synthesized via tryptophan in the liver in animals and glycerol and aspartic acid as precursors in plants, respectively, and quinolinic acid. It is also synthesized from the vitamins nicotinic acid and nicotinamide. NAD ⁺ important feature lies in the reduction of NAD ⁺ to the biological energy production mechanism (oxidation reaction) are conjugated.

NADP is a coenzyme that acts on glucose-6-phosphate dehydrogenase. The structure of NADP is basically the same as that of NAD, and is a structure in which phosphoric acid is further ester-bonded to the 2 ′ position of ribose of adenylic acid of NAD. NADP is abundant in the liver in vivo, but its content is about half that of NAD. The conversion mode of the oxidized and reduced forms of NADP is the same as that of NAD, and oxidized NADP has a positive (+) charge due to pyridinium ions (NADP ⁺ ). Although NADP and NAD are similar in structure and reaction mode, the enzyme strictly distinguishes them. Reactions involving NADP include glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, L-glutamate dehydrogenase, and the like.

  Examples of the ATP-dependent NAD kinase containing the GGDGN motif include those derived from γ-proteobacteria and homologs thereof. For example, Escherichia bacterium, Shigella genus bacteria, Salmonella genus bacteria, Klebsiella genus bacteria, Proteus genus bacteria, Serratia genus bacteria, Yersinia genus bacteria, Erwinia genus bacteria, Photohabdus genus bacteria, Wiggleshorthus bacterium, Haemophilus genus bacteria, Mannheimia genus bacteria, Pasteurella genus bacteria, Aeromonas genus bacteria, Vibrio genus bacteria, Photobacterium bacteria, Shewanella genus bacteria, Colwellia genus bacteria, Idiomarina genus bacteria, Pseudobacteria genus Those derived from, and the like.

  Specifically, Escherichia coli, Shigella boydii, Shigella flexneri, Salmonella enterica, Klebsiella pneumoniae, Yersinia pestis, Erwinia carofovora, Photorhabdus luminescens, Wigglesworthia glossinidia, Buchnera aphidicola, Sodalis glossinidius, Haemophilus influenzae, Mannheimia succiniciproducens, Pasteurella multocida, Aeromonas hydrophila, Aeromonas salmo icida, Vibrio cholerae, Vibrio fischeri, Photobacterium profundum, Shewanella oneidensis, Colwellia psychrerythraea, Idiomarina loihiensis, Pseudoalteromonas haloplanktis, Cronobacter sakazakii, those derived from the Dickeya dadantii.

  Examples of ATP-dependent NAD kinase containing GGDGN motif, ecoNADK (Escherichia coli), styNADK (Salmonella enterica), vchNADK (Vibrio cholerae), sfl_SF2674K (Shigella flexneri), kpn_KPN_02937 (Klebsiella pneumoniae), esa_ESA_00641 (Cronobacter sakazakii), And dda_Dd703_3217 (Dickeya danantii). The amino acid sequences of the above ecoNADK, styNADK, vchNADK, sfl_SF2674K, kpn_KPN_02937, esa_ESA_00641 and dda_Dd703_3217 are represented by SEQ ID NOs: 1 to 7, respectively. The amino acid sequence of a known NAD kinase and the base sequence of the gene encoding it can be obtained from the NCBI database (www.ncbi.nlm.nih.gov), for example.

  Examples of the ATP-dependent NAD kinase that can confer polyphosphate utilization in the present invention include a protein (NAD kinase) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 7. A protein functionally equivalent to the protein consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7, for example, 70% or more, preferably 80% or more, more preferably 90% to any of the amino acid sequences of SEQ ID NOs: 1 to 7 % Or more, more preferably 95% or more, particularly preferably 99% or more, the present invention is also applied to a protein comprising an amino acid sequence carrying a GGDGN motif and having an activity of an ATP-dependent NAD kinase it can. Furthermore, in the amino acid sequence of any one of SEQ ID NOs: 1 to 7, it comprises an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added but the GGDGN motif is retained, and the ATP-dependent NAD kinase The present invention can also be applied to proteins having activity. The term “several” means 2 to 5, preferably 2 to 3.

The GGDGN motif is a partial sequence consisting of “Gly-Gly-Asp-Gly-Asn” in the amino acid sequence encoding an ATP-dependent kinase, and the present inventors have identified the ATP-dependent kinase, particularly the primary structure of the ATP-dependent NAD kinase. And found that this GGDGN motif is highly conserved and involved in its catalytic action. For example, in the amino acid sequence of SEQ ID NO: 1 encoding ecoNADK, the GGDGN motif is located at positions ^{71 to 75} ( ⁷¹ GGDGN ⁷⁵ ).

  The present invention, in an ATP-dependent kinase containing a GGDGN motif, confers polyphosphate utilization to an ATP-dependent kinase by replacing the Asn residue (N) of the GGDGN motif with a Thr residue (T). Thereby, a kinase mutant having the ability to use polyphosphate is obtained. In the present invention, imparting polyphosphoric acid utilization ability includes increasing polyphosphoric acid utilization ability. Increasing the availability of polyphosphate refers to an increase in the specific activity of the kinase mutant for polyphosphate as compared to the specific activity of wild-type kinase for polyphosphate. Preferably, in an ATP-dependent NAD kinase containing a GGDGN motif, by replacing the Asn residue (N) of the GGDGN motif with a Thr residue (T), the ATP-dependent NAD kinase is imparted with the ability to use polyphosphate, thereby A NAD kinase mutant having polyphosphate utilization ability is obtained.

  The Asn residue of the GGDGN motif to be substituted is preferably an Asn residue at a position corresponding to position 75 when SEQ ID NO: 1 is used as a reference sequence. In the present specification, the expression “at a position corresponding to the X position when SEQ ID NO: 1 is used as a reference sequence” is used in the amino acid sequence of any ATP-dependent kinase having a GGDGN motif with SEQ ID NO: 1 as a reference sequence. Used to specify the position of a given amino acid residue. That is, the amino acid sequence of an arbitrary ATP-dependent kinase having a GGDGN motif (hereinafter referred to as amino acid sequence Y) is counted from the first amino acid residue of SEQ ID NO: 1 when aligned with the amino acid sequence of SEQ ID NO: 1. Means an amino acid residue in amino acid sequence Y that is aligned to the Xth amino acid residue (ie, aligned in the same column in the alignment). The positions of the amino acid residues are in particular BLAST (Altschul, SF, J. Mol. Biol. 215, 403-410 (1990)) and CLUSTALW (Thompson, JD, Nucleic. Acids) as alignment software. Res.22, 4673-4680 (1994)) means the position of an amino acid residue. These programs are available from GenomeNet server (http://www.genome.jp).

  In the amino acid sequence of SEQ ID NO: 2 encoding styNADK, the GGDGN motif is located at positions 71 to 75, and the Asn residue to be substituted is located at position 75. In the amino acid sequence of SEQ ID NO: 3 encoding vchNADK, the GGDGN motif is located at positions 72 to 76, and the Asn residue to be substituted is located at position 76. In the amino acid sequence of SEQ ID NO: 4 encoding sfl_SF2674K, the GGDGN motif is located at positions 71 to 75, and the Asn residue to be substituted is located at position 75. In the amino acid sequence of SEQ ID NO: 5 encoding kpn_KPN_02937, the GGDGN motif is located at positions 71 to 75, and the Asn residue to be substituted is located at position 75. In the amino acid sequence of SEQ ID NO: 6 encoding esa_ESA_00641, the GGDGN motif is located at positions 71 to 75, and the Asn residue to be substituted is located at position 75. In the amino acid sequence of SEQ ID NO: 7 encoding dda_Dd703_3217, the GGDGN motif is located at positions 71 to 75, and the Asn residue to be substituted is located at position 75.

  In the amino acid sequence of an ATP-dependent kinase, as a method for obtaining a kinase mutant by substituting the Asn residue of the GGDGN motif with a Thr residue, a method known in the art can be used, a method by chemical synthesis, and genetic engineering. In the technique using, a method of changing a codon of a base sequence encoding an amino acid sequence is included, but is not limited thereto. In the present invention, a variant has an amino acid sequence in which at least one amino acid in the amino acid sequence of the original enzyme is substituted, added, deleted, or modified, and at least a part of the activity of the original enzyme. A modified enzyme that retains.

  The kinase mutant of the present invention is obtained by, for example, introducing a mutation into a wild type gene encoding an ATP-dependent kinase, cloning the obtained mutant gene into a cloning vector, and transforming a host cell with the obtained recombinant vector. The transformed host cell can be cultured in an appropriate medium, and finally, the obtained kinase mutant can be isolated and purified.

The introduction of a mutation into a gene is not particularly limited, but can be performed using a known in vitro mutation technique such as a site-directed mutagenesis method. For example, annealing a primer containing a mutation to a recombinant plasmid containing a wild type gene, then synthesizing DNA containing the mutation in vitro by allowing DNA polymerase to act, and finally transforming the host cell Can be used to isolate clones containing mutations (Kunkel, TA (1985) Proc. Natl. Acad. Sci. USA 82, 488 .; Hashimoto-Gotoh, T. et al. (1995) Gene. 152, 271 .; Weiner MP, et al. (1994) Gene.Dec 30; 151 (1-2): 119-23.; WO97 / 20950). A commercially available site-directed mutagenesis kit may be used. Examples of such kits include KOD-Plus-Mutageness kit (Toyobo Co., Ltd.), Quick-change kit (Strategene), and Transformer ^™ Site-Directed Mutagenesis kit.

Preparation of a genomic DNA library used for cloning, hybridization, PCR, preparation of plasmid DNA, cleavage and ligation of DNA, transformation, etc. are performed by well-known gene recombination techniques (for example, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor). Laboratory ^Press, referring to 4 th Edition (2012)) can be carried out employing appropriately.

  The kinase mutant having the ability to use polyphosphate obtained by the above method, preferably the NAD kinase mutant, has an amino acid sequence containing a GGDGN motif encoding an ATP-dependent kinase, wherein the Asn residue of the GGDGN motif is replaced with a Thr residue. It has a polyamino acid utilization ability.

  The NAD kinase mutant having the ability to use polyphosphate obtained in the present invention includes an amino acid sequence in which the Asn residue of the GGDGN motif is substituted with a Thr residue in any one of the amino acid sequences of SEQ ID NOs: 1 to 7, ie, a sequence What consists of protein which consists of an amino acid sequence in any one of Nos. 8-14 is mentioned. The protein comprising any one of the amino acid sequences of SEQ ID NOs: 8 to 14 has a GGDGT motif in which the Asn residue of the GGDGN motif is replaced with a Thr residue. That is, the GGDGT motif is a partial sequence consisting of “Gly-Gly-Asp-Gly-Thr”. A protein functionally equivalent to a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 8 to 14, for example, 70% or more, preferably 80% or more, more preferably 90%, of any of the amino acid sequences of SEQ ID NOs: 8 to 14 % Or more, more preferably 95% or more, particularly preferably 99% or more, a protein comprising an amino acid sequence carrying a GGDGT motif, having polyphosphate utilization ability, and having NAD kinase activity Are encompassed by the kinase variants of the present invention. Furthermore, the amino acid sequence of any one of SEQ ID NOs: 8 to 14 comprises an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added but the GGDGT motif is retained. A protein having NAD kinase activity is also included in the kinase variant of the present invention. The term “several” refers to 2 to 30, preferably 2 to 5, more preferably 2 to 3.

  The kinase having the ability to use polyphosphate refers to a kinase having the ability to use polyphosphate as a phosphate donor in the phosphorylation reaction. What uses both polyphosphoric acid and ATP as a phosphate supply substrate is also included.

  Polyphosphoric acid is a polymer of an inorganic orthophosphoric acid residue via a phosphoric anhydride bond that is energetically equivalent to the phosphate bond of ATP, and is represented by the following formula, for example.

  In the above formula, n represents the degree of condensation. Although n is not particularly limited, n is preferably 3 to 5000, and more preferably 3 to 32. Polyphosphoric acid is commercially available in large quantities at a very low price compared to ATP. Specific examples of polyphosphoric acid include tripolyphosphoric acid (phosphoric acid residue number: 3), tetrapolyphosphoric acid (phosphoric acid residue number: 4), trimetaphosphoric acid (cyclic polyphosphoric acid, phosphoric acid residue number: 3), hexametalin. Examples include acids (long-chain polyphosphoric acid mixtures) and metaphosphoric acids (long-chain polyphosphoric acid mixtures). The polyphosphoric acid includes salts of polyphosphoric acid as described above. Polyphosphoric acid is available from, for example, Wako Pure Chemical Industries, Sigma-Aldrich, and Merck.

Metaphosphoric acid is also called icy phosphoric acid and is represented by the general formula (HPO ₃ ) _n . Usually, it exists as a polymer such as trimer, tetramer cyclic or linear polymetaphosphoric acid. In this specification, metaphosphoric acid includes such trimer, tetramer cyclic or linear polymetaphosphoric acid. Polymetaphosphoric acid is a compound in which phosphoric acid groups are linked cyclically by acid anhydride bonds, and is represented by the general formula H _n P _n O _3n . It is a viscous liquid at room temperature and becomes a glassy solid solution upon cooling. The aqueous solution is acidic and easily decomposes into normal phosphoric acid when heated. It is hydrolyzed by metaphosphatase to open the ring and become polyphosphate. In the biological world, high molecular weight compounds are found in bacteria, fungi, algae, etc., and yeast and certain bacteria also have a low degree of condensation such as trimetaphosphoric acid. The phosphoric anhydride bond is a so-called high energy phosphate bond.

  The NAD kinase mutant having the ability to utilize polyphosphate can catalyze the following phosphorylation reaction that produces NADP from NAD using polyphosphate as a phosphate donor.

  The kinase variant having the ability to use polyphosphate of the present invention preferably has a reaction rate with respect to polyphosphate of 40% or more, more preferably 60% or more, still more preferably 120% or more, most preferably 150% of the reaction rate with ATP. That's it. In the kinase variant of the present invention, the specific activity against polyphosphate is preferably 1.5 times, more preferably 2 times, and even more preferably 3 times or more, compared to the specific activity of wild-type kinases against polyphosphate. doing.

  The present invention also relates to a method for producing nicotinamide adenine nucleotide phosphate (NADP), which comprises polyphosphate or a salt thereof and nicotinamide adenine dinucleotide (NAD) as a substrate and phosphorylation using the NAD kinase mutant. Performing an oxidation reaction.

  Various conditions such as temperature, pH, type of polyphosphoric acid, concentration of polyphosphoric acid in the reaction solution, concentration of metal ion, type of metal ion, etc. can be appropriately determined by those skilled in the art. The concentration of polyphosphoric acid or a salt thereof in the reaction solution is 0.1 to 15% by weight of the reaction solution. The kind of polyphosphoric acid is not specifically limited. For example, it can be appropriately selected from the previously described metaphosphoric acid, hexametaphosphoric acid and the like. In the above reaction of the NAD kinase mutant, it is preferable that a metal ion, particularly a divalent metal ion, is present. The concentration of the divalent metal ion in the reaction solution is 5 to 150 mM. The divalent metal ion is not particularly limited, but is preferably selected from magnesium ions or manganese ions. Particularly preferred is magnesium ion. The metal ions are preferably included in the reaction solution as chlorides, sulfates or nitrates.

  Furthermore, in order to obtain NADP in a high yield after completion of the reaction, it is important to keep the NADP once synthesized in addition to the high phosphate transfer efficiency from NAD. NADP is known to be more unstable to heat and pH than NAD (for example, the Oriental Yeast 2000 Catalog). Therefore, it is preferable to carry out the reaction conditions with a composition in which NADP is stable. Although not necessarily limited, as reaction conditions, the temperature is preferably 20 to 37 ° C, more preferably 37 ° C. The pH is preferably 5 to 8, more preferably 6 to 7.

  The form of the NAD kinase mutant used in the present invention may be a solubilized protein purified from a recombinant host cell, or an immobilized enzyme on which the solubilized protein is immobilized. Alternatively, an immobilized bacterial enzyme in which a bacterial cell expressing a NAD kinase mutant is directly immobilized by chemical treatment such as acrylamide may be used. The enzyme immobilization method can be performed using a known method. For example, the purified NAD kinase can be mixed with the activated gel under mild conditions that do not inactivate and suspended to immobilize the purified NAD kinase on the insolubilized gel. Known methods for immobilizing microbial cells include methods using carrageenic acid or acrylamide.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to the range of an Example.

Example 1 Homology and Sequence Alignment Analysis To identify proteins homologous to NAD kinase (NADK) for all species whose genomic sequence has been determined and to confirm the amino acid residue corresponding to Asa-75 of ecoNADK BLAST and CLUSTALW were used.

  FIG. 1 shows alignments of primary structures of ATP-NADK, polyphosphate / ATP-NADK and γ-proteobacterial NADK homolog (SEQ ID NO: 1 to 7 and SEQ ID NO: 15 to 21 in order from the top). ATP-NADK refers to an ATP-dependent NAD kinase, that is, an NAD kinase that substantially utilizes only ATP as a phosphate donor, and polyphosphate / ATP-NADK serves as both a phosphate and ATP as a phosphate donor. Refers to NAD kinase that utilizes

  ecoNADK (Escherichia coli), styNADK (Salmonella enterica), vchNADK (Vibrio cholerae), sph_NADK (Sphingomonas sp. A1), hsa_65c (es), and scaYc49. Ppnk (Mycobacterium tuberculosis), bsu_BSU11610 (Bacillus subtilis), mja_MJ_0917 (Methanocaldococcus jannaschii), and pho_PH1074 (Pyrococcus hor). ecoNADK, styNADK, vchNADK, sfl_SF2674K (Shigella flexneri), kpn_KPN_02937 (Klebsiella pneumoniae), esa_ESA_00641 (Cronacter sakadad)

EcoNADK Asn-75 is indicated by an arrow. The residue corresponding to Asn-75 is conserved as an Asn residue in the γ-proteobacterial NADK homolog, but is a Thr residue in other ATP-NADK and polyphosphate / ATP-NADK. Asn-75 adjacent to the ⁷¹ GGDG ⁷⁴ motif of ecoNADK is conserved as GGDGN in the primary structure of the gamma proteobacterial NADK homolog containing styNADK and vchNADK. On the other hand, in other ATP-NADK and polyphosphate / ATP-NADK, Asn-75 corresponds to Thr and forms a GGDGT motif. The GGDG motif is highly conserved in the primary structure of NADK and is thought to be involved in its catalytic action.

(Example 2) Construction of NADK mutant In Example 1, since the GGDGN motif is highly conserved in the NADK homologue of γ-proteobacteria, in order to elucidate the function of this GGDGN motif, Asn-75 of ecoNADK And a variant in which Asn-76 of vchNADK was replaced with a Thr residue. Moreover, the mutant which substituted Thr-87 of Ppnk (Polyphosphate-dependent NAD kinase) with the Asn residue was also constructed. In addition, various ecoNADK mutants were constructed (ecoNADK E36A, E163V, R232D, L259E, E36A / E136V, R232D / L259E, E36A / E136V / R232D / L259E, R95L, N97R, R95L / N97R, and mutant 4). Variant 4 is ecoNADK E36A / E136V / R232D / L259E.

First, ecoNADK (SEQ ID NO: 1), vchNADK (SEQ ID NO: 3 ) and Ppnk (SEQ ID NO: 18) were cloned into pET-14b or pET-3a. Various NADK mutants were constructed using KOD-Plus-Mutageness kit (Toyobo). However, KOD-Plus-Neo polymerase (Toyobo) was used as the polymerase. Plasmids pET-14b_ecoNADK, pET-14b_vchNADK, or pET-3a_Ppnk were used as PCR templates. The base sequence of the obtained gene was confirmed by DNA sequencing. Wild-type or mutant NADKs are all E. coli without His-tag. expressed in E. coli BL21 (DE3) pLysS (Novagen) and purified as described in the literature (Kawai, S. et al., Eur. J. Biochem. 268, 4359-4365 (2001); Kawai, S. et al. Biochem. Biophys. Res. Commun. 276, 57-63 (2000)). However, KNDE buffer [10 mM KPB (pH 7.0), 0.1 mM NAD ⁺ , 0.5 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid] was used in place of the KND buffer.

(Example 3) Assay of NADK activity The ATP-dependent NADK activity or polyphosphate-dependent NADK activity of wild-type or mutant NADK prepared in Example 2 was continuously assayed at 37 ° C by the method described in the literature ( Ohashi, K., et al., Mol. Cell. Biochem. 355, 57-64 (2011)). However, 100 mM Tris-HCl (pH 7.5) was used instead of 100 mM Tris-HCl (pH 8.0).

Specifically, 5.0 mM NAD, 5.0 mM ATP, 5.0 mM glucose-6-phosphate, 0.5 U G6P dehydrogenase (G6PDH) (Sigma, St. Louis, MO), 5.0 mM MgCl ₂ , 100 mM 1 ml of a reaction solution containing Tris-HCl buffer (pH 7.5) and an appropriate amount of NADK was kept at 37 ° C., and the amount of NADPH produced was continuously monitored at 340 nm. When measuring polyphosphate-dependent activity, 5 mM tripolyphosphate or tetrapolyphosphate, 1 mg / m1 hexametaphosphate or metaphosphate was used instead of ATP. The amount of enzyme capable of producing 1 μmol of NADP (H) in 1 minute was defined as 1 unit (U).

When Asn-75 was replaced with Thr in ecoNADK, the specific activity against ATP (U / mg) decreased from 28 to 7.4, and among the polyphosphates, the specific activity against tetrapolyphosphate (Poly (P) ₄ ) was 0. The specific activity against hexametaphosphoric acid (hexameta (P) _n ) increased from 0.85 to 3.6 and the specific activity against metaphosphoric acid (meta (P) ₄ ) was 0.8. Increased to 3.2 (Fig. 2a). The specific activity (U / mg) of the vchNADK mutant also showed a similar behavior, the specific activity against ATP (U / mg) decreased from 11 to 5.1, and the specific activity against tripolyphosphate (Poly (P) ₃ ) was Increased from 0.076 to 0.50, specific activity for tetrapolyphosphoric acid increased from 0.69 to 3.0, specific activity for hexametaphosphoric acid increased from 1.1 to 7.3, ratio to metaphosphoric acid The activity increased from 1.2 to 7.8 (Figure 2a). Hexametaphosphoric acid and metaphosphoric acid are a mixture of polyphosphoric acids having different chain lengths.

  The ratio (%) of polyphosphate-dependent NADK activity to ATP-dependent NADK activity of ecoNADK and vchNADK increased from 2.8 to 43 by substitution (ecoNADK) and from 11 to 154, respectively (vchNADK) (FIG. 2b). ) And other substitutions did not increase (FIG. 3). On the other hand, Ppnk T87N had reduced both ATP-dependent NADK activity and polyphosphate-dependent NADK activity compared to the wild type (FIG. 2).

  From these results, the Asn residue (Asn-75 of ecoNADK or Asn-76 of vchNADK) is the residue that determines the specificity for ATP, and the Thr residue at this position is at least ecoNADK and vchNADK, and GGDGN It can be seen that it is important for polyphosphate utilization in other γ-proteobacterial NADK homologs with motifs.

The NADK reaction mixture (5 μl) was incubated at 37 ° C. and analyzed by TLC. TLC analysis was performed by detecting at 254 nm using isobutyrate and 500 mM NH ₄ OH as the solvent system at 5: 3 (v / v) (Kawai, S. et al., J. Basic Microbiol. 44, 185-196 (2004)). The composition of the reaction mixture is as follows: 5 mM NAD ⁺ , 5 mM MgCl ₂ , 100 mM Tris-HCl (pH 7.5), 5 mM ATP or 1.0 mg / ml metaphosphate, and 0.43 μg / ml ecoNADK or ecoNADK N75T. As a control, the following reaction mixture (5 μl) was used: 5 mM NAD ⁺ , 5 mM NADP ⁺ , 5 mM ATP, and 5 mM ADP. The results are shown in FIG.

TLC analysis confirmed that ecoNADK N75T produced more NADP ⁺ than wild type when metaphosphate was used as the phosphate donor (FIG. 4).

In addition, the k _cat value for tetrapolyphosphate of each enzyme was increased by this single substitution (Table 1).

  From the above, it was found that ecoNADK N75T and vchNADK N76T acquired polyphosphate availability instead of losing specificity for ATP as a phosphate donor. This polyphosphate availability was comparable to or higher than that of Ppnk (FIGS. 2a and 2b, Table 1). This is the first time that ATP-dependent kinase has been successfully bioengineered to impart polyphosphate utilization.

  The present invention is expected to enable large-scale production of higher-purity useful phosphorylated substances (NADP, diagnostic agents, reagents, pharmaceuticals, intermediates of various compounds, etc.) by enzymatic methods at lower cost. Is done.

Claims (6)

A method for imparting polyphosphate utilization to an ATP-dependent kinase, comprising:
In the ATP-dependent kinase consisting of the amino acid sequence of SEQ ID NO: 1 or 3 containing the GGDGN motif, the method comprises replacing the Asn residue of the GGDGN motif with a Thr residue.   2. The method of claim 1, wherein the ATP dependent kinase is an ATP dependent NAD kinase.   The method according to claim 1 or 2, wherein the ATP-dependent kinase is derived from γ-proteobacteria.   The method according to any one of claims 1 to 3, wherein the Asn residue of the GGDGN motif is an Asn residue at a position corresponding to position 75 when SEQ ID NO: 1 is used as a reference sequence. A method of imparting polyphosphate utilization to a protein having an activity of phosphorylating NAD using ATP,
A protein having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 or 3 , comprising an amino acid sequence containing a GGDGN motif, and having an activity of phosphorylating NAD using ATP, or of SEQ ID NO: 1 or 3 A GGDGN motif in a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added, but including a GGDGN motif, and having an activity to phosphorylate NAD using ATP Substituting a Thr residue for the Asn residue. A kinase variant comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 8 or 10 (b) comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 8 or 10 and retaining the GGDGT motif; Protein having activity of phosphorylating NAD using (c) In the amino acid sequence of SEQ ID NO: 8 or 10, one or several amino acids are deleted, substituted, inserted or added, but the GGDGT motif is retained A protein comprising an amino acid sequence and having an activity of phosphorylating NAD using polyphosphate.

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