Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fukuda, C. Articles by Murata, K. Search for Related Content PubMed PubMed Citation Articles by Fukuda, C. Articles by Murata, K. Agricola Articles by Fukuda, C. Articles by Murata, K.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2007, p. 5447-5452, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.02703-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

NADP(H) Phosphatase Activities of Archaeal Inositol Monophosphatase and Eubacterial 3'-Phosphoadenosine 5'-Phosphate Phosphatase

Chikako Fukuda, Shigeyuki Kawai, and Kousaku Murata^*

Department of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received 20 November 2006/ Accepted 25 June 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP⁺ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3'-phosphoadenosine 5'-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3'-phosphoadenosine 5'-phosphate phosphatase. Based on this fact, we found that 3'-phosphoadenosine 5'-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3'-phosphoadenosine 5'-phosphate phosphatase rather than as NADP(H) phosphatase.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
NADP(H) (NADP⁺ and NADPH) phosphatase [NADP(H)ase] converts NADP(H) into NAD(H) (NAD⁺ and NADH), i.e., NADP(H)ase dephosphorylates the 2'-phosphate group on the adenosine moiety of NADP(H), and has been proposed to regulate the intracellular NAD(H)/NADP(H) balance in concert with NAD kinase (9, 14, 25). NAD kinase catalyzes the formation of essential NADP(H) through the phosphorylation of NAD(H). Although NADP(H)ase has not been identified in eukaryotes and eubacteria, it appears to exist in the eubacterium Escherichia coli since it has been estimated that 140 NADP⁺ molecules are degraded to NAD⁺ by NADP(H)ase per second in an average E. coli cell in the exponential growth phase (14).

Inositol monophosphatase (IMPase) catalyzes the dephosphorylation of D-myo-inositol monophosphate (IMP) to yield myo-inositol (2). Fructose-1,6-bisphosphatase (FBPase) catalyzes the dephosphorylation of 1-phosphate on fructose-1,6-bisphosphate (FBP) to yield fructose-6-phosphate (18) and is a key enzyme in gluconeogenesis (21). IMPase and FBPase exist as two distinct gene products in eukaryotes and some eubacteria and are specific to IMP and FBP, respectively (26). However, the IMPase homologues in some hyperthermophilic organisms (archaea and eubacteria) exhibit both IMPase and FBPase activities (3, 19, 23, 24). The tertiary structure of archaeal IMPase resembles those of eukaryotic IMPase and FBPase (7, 23, 24), 3'-phosphoadenosine 5'-phosphate (3'-PAP [Fig. 1]) phosphatase (3'-PAPase), and mammalian inositol polyphosphate 1-phosphatase (IPPase) (13, 22, 24). The enzyme 3'-PAPase removes the 3'-phosphate group on 3'-PAP to yield 5'-AMP (Fig. 1), although the IPPase and 3'-PAPase activities of archaeal IMPases have not been reported. These enzymes share a common motif that corresponds to a metal-binding site (26) and form an extended protein family designated the "IMPase/FBPase-like family" in the Structural Classification of Proteins (SCOP) database (1).

View larger version (12K): [in this window] [in a new window]

FIG. 1. Structure of NADP+ and its analogues. "P" represents the phosphoryl group. The arrow in NADP+ indicates the position at which the MazG nucleotide pyrophosphatase domain and the NUDIX protein could attack to cleave the pyrophosphate moiety.

Recently, a novel relationship between NADP(H)ase and archaeal IMPase has been established based on our findings regarding MJ0917 of the hyperthermophilic methanogenic archaeon Methanococcus jannaschii (8). MJ0917 is a fusion protein comprising an IMPase homologue and NAD kinase. The IMPase homologous region in MJ0917 exhibits activities of FBPase and NADP(H)ase but not IMPase (8). Furthermore, in some archaea (Methanobacterium thermoautotrophicum, Methanosarcina acetivorans, Methanosarcina mazei, Archaeoglobus fulgidus, and Methanopyrus kandleri), IMPase homologues form operon-like structures, together with NAD kinase homologues in their genome sequences, implying a further relationship of archaeal IMPase homologues with NADP(H)ase (8).

In this study, we demonstrated that the two hyperthermophilic archaeal IMPases, MJ0109 of M. jannaschii and AF2372 of A. fulgidus, also exhibit NADP(H)ase and IPPase activities. Furthermore, the structural relationship of archaeal IMPase with the proteins belonging to the IMPase/FBPase-like family led to our next finding that E. coli CysQ belonging to this family exhibits NADP(H)ase activity.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Assays.
The phosphatase activities of MJ0109, AF2372, and MJ0917 were assayed at 85°C in 0.10 ml of a mixture containing 1.0 mM phosphorylated compound, 20 mM MgCl₂, and 100 mM Tris-HCl (pH 8.5 at 85°C) (8). The phosphatase activity of CysQ was assayed at 37°C in 0.10 ml of a mixture containing 1.0 mM phosphorylated compound, 10 mM MgCl₂, and 100 mM Tris-HCl (pH 8.0 at 37°C), unless otherwise stated. The optimum pH for CysQ was determined by using 100 mM Tris-HCl (pH 6.7 to 8.9) and 100 mM Tris-maleate (pH 5.9 to 7.1). Phosphatase activities of E. coli cell extracts were assayed at 37°C in 0.10 ml of a mixture containing 0.50 mM phosphorylated compound, 5.0 mM MgCl₂, and 100 mM Tris-HCl (pH 8.0 at 37°C). The reaction was stopped by the addition of 0.10 ml of 10% sodium dodecyl sulfate (SDS) (6, 9), and the amount of released P_i was determined as described previously (5). In the presence of ATP, the amount of P_i was assayed using an alternative method (6). The nonenzymatic release of P_i was subtracted from the total P_i released. Less than 10 µl of a purified enzyme solution (for MJ0109, 0.39 mg/ml; for AF2372, 0.05 mg/ml; for MJ0917, 0.014 mg/ml; or for CysQ, 0.54 to 0.027 mg/ml) was routinely added to the reaction mixture. In thermophilic enzyme assays, the reactions were initiated by adding the enzyme immediately after the reaction mixtures had been preincubated at 85°C for 3 min. In the presence of LiCl, the activities were assayed in the presence of substrate at concentrations close to the K_m value. For AF2372, the concentrations of NADP⁺, NADPH, FBP, and IMP were 0.15, 0.4, 0.1, and 0.2 mM, respectively; for MJ0109, the concentrations of NADP⁺, NADPH, FBP, and IMP were 0.5, 0.7, 0.7, and 1.0 mM, respectively; and for CysQ, the concentrations of NADP⁺, NADPH, FBP, 3'-PAP, and 2'-PAP were 2.5, 2.0, 1.2, 0.3, and 0.2 mM, respectively. The assays were linear with respect to time (at least 2 points) and concentrations of enzyme preparations (at least 2 points). One unit of enzyme activity was defined as 1.0 µmol of product formed in 1 min at 85°C or 37°C, and the specific activity was expressed as units per milligram of protein. In the purification procedure, the protein concentrations were determined by using the Bradford reagent (Sigma, St. Louis, MO) with bovine serum albumin as a standard. The protein concentrations of the purified MJ0109, AF2372, and CysQ were determined from the extinction coefficient (₂₈₀) (for MJ0109, 30,700 [4]; for AF2372, 16,390; and for CysQ, 44,920) by using equations obtained from ExPASy (http://us.expasy.org/tools/protparam.html). The K_m and k_cat values were calculated using a Hanes-Woolf plot.

Cloning and expression.
The genes encoding MJ0109 and AF2372 were amplified by PCR by using genomic DNA of M. jannaschii and A. fulgidus purchased from the American Type Culture Collection (43067D and 49558D, respectively) as templates and inserted into the NdeI/XhoI sites of pET-21b (Novagen, Darmstadt, Germany). The authenticity of the cloned genes was confirmed by DNA sequencing. The resultant plasmids, MJ0109-pET-21b and AF2372-pET-21b, were introduced into E. coli BL21(DE3) (Novagen) and E. coli Origami B(DE3) (Novagen), resulting in strains MK1087 and MK1532, respectively. The strains were cultivated in Luria-Bertani (LB) medium (0.5% yeast extract, 1.0% tryptone, and 1.0% NaCl, pH 7.2) containing 100 µg/ml ampicillin (13.5 liters for MK1087 and 10.5 liters for MK1532) according to the pET system manual (Novagen), except for cultivation times of 44 h for MK1087 and 60 h for MK1532 at 18°C, following the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) (1 mM final concentration) to the cultures having an A₆₀₀ of approximately 0.80.

To express SuhB, Fbp, and CysQ, E. coli K-12 AG1 ASKA green fluorescent protein (GFP)-free strains, which contained the plasmid pCA24N carrying each of the suhB, fbp, and cysQ genes (10), were cultivated at 37°C in LB medium containing 30 µg/ml chloramphenicol to an A₆₀₀ of 0.8. After adding IPTG to a 0.1-mM final concentration, the strains were further cultivated at 37°C for 5.5 h. The suhB, fbp, and cysQ genes were expressed as proteins attached to six histidine residues and seven spacer amino acid residues (Thr-Asp-Pro-Ala-Leu-Arg-Ala) at the N-terminal end and five additional residues (Gly-Leu-Cys-Gly-Arg) without GFP at the C-terminal end (10). As a control, the E. coli K-12 AG1 strain carrying pCA24N alone was treated in the same manner.

The E. coli cells were harvested by centrifugation and washed once with Tris-EDTA (TE) (10 mM Tris-HCl, pH 8.5, at 4°C and 1.0 mM EDTA). The cells were suspended in TE and then disrupted by sonication at 4°C using an Insonator 201 M sonicator (Kubota, Tokyo, Japan). The clear supernatant, which was obtained by centrifugation at 20,000 x g for 10 min at 4°C, was used as a cell extract.

Purification.
The MJ0109 protein was purified from the MK1087 cells by following a previously reported procedure (4, 7). Briefly, the cell extract (4,312 mg in TE) of the MK1087 cells (84 g wet weight) expressing MJ0109 was heated for 30 min at 85°C. After centrifugation at 20,000 x g for 10 min, the supernatant was applied to a Toyopearl Super Q-650S (TOSOH, Tokyo, Japan) column (2.5 by 16 cm). The MJ0109 protein was eluted with a gradient of KCl (0 to 500 mM, 230 ml) in TE. After the addition of NaCl to the active solution (obtained at approximately 200 mM KCl) to attain a final concentration of 500 mM, the solution was applied to a phenyl-Sepharose (Amersham Biosciences, Piscataway, NJ) column (2.5 by 8 cm). The MJ0109 protein was eluted with a gradient of NaCl (500 to 0 mM, 120 ml) in TE and then TE alone (500 ml). The active solution (520 ml) was concentrated to approximately 5 ml by using an Amicon ultrafiltration cell (Amicon, Beverly, MA) equipped with a membrane with a nominal molecular weight limit of 10,000 and applied to a Sephacryl S-200 (Amersham Biosciences) column (2.7 by 56 cm). The MJ0109 protein was eluted with 20 mM Tris-HCl (pH 8.5 at 4°C) and used as the purified MJ0109.

The AF2372 protein was purified from the MK1532 cells as described previously (24). Briefly, the cell extract (4,312 mg in TE) of the MK1532 cells (51 g wet weight) expressing AF2372 was treated using the same procedure described for the purification of MJ0109, with the exception that a Toyopearl Super Q-650S column with different dimensions (2.5 by 20 cm) was used. No phenyl-Sepharose column was used, and the AF2372 protein was eluted from a Sephacryl S-200 column by using 20 mM Tris-HCl (pH 8.5 at 4°C) containing 1.0 mM EDTA, as described previously (24). The eluted protein was used as the purified AF2372. Purified MJ0917 was obtained as described previously (8).

For the purification of CysQ, an extract (212 mg protein in 26.5 ml HEPES-sodium chloride [HS] [20 mM HEPES, pH 7.0, and 300 mM NaCl]) of E. coli cells expressing histidine-tagged CysQ protein from the cysQ gene in pCA24N was obtained from a 1.4-liter culture, as described above. The cell extract was applied to a TALON metal affinity resin column (2.0 by 9.0 cm) (Clontech, Mountain View, CA) equilibrated with HS. The histidine-tagged CysQ protein was purified according to the protocol supplied by Clontech. Briefly, after the column was washed with 125 ml HS and with 50 ml HS containing 10 mM imidazole, the CysQ protein was eluted with HS containing 150 mM imidazole. The fraction containing the purified CysQ was mixed with an equal volume of 50% glycerol and used as the purified CysQ protein. For the assay, 5 or 10 µl of the purified CysQ solution diluted with a stabilizing buffer (10 mM HEPES [pH 7.0], 150 mM NaCl, 75 mM imidazole, and 25% glycerol) was used; this resulted in contamination of the reaction mixture with the following components: 15 mM NaCl, 7.5 mM imidazole, and 2.5% glycerol. These components, however, had no effect on the 3'-PAPase, NADP(H)ase, and FBPase activities. This was confirmed by using the CysQ solutions diluted with the stabilizing buffer or 10 mM HEPES (pH 7.0) alone.

Other analytical methods.
SDS-polyacrylamide gel electrophoresis (PAGE) was conducted using a 12.5% gel, as described elsewhere (12). The proteins in the gel were visualized using Coomassie brilliant blue R-250.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
NADP(H)ase activities of MJ0109 and AF2372.
We selected two hyperthermophilic archaeal IMPases, MJ0109 of M. jannaschii and AF2372 of A. fulgidus. MJ0109 is an IMPase homologue distinct from MJ0917 that is found in the genomic sequence of M. jannaschii, and AF2372 is the sole IMPase homologue found in the genomic sequence of A. fulgidus. MJ0109 and AF2372 are known to exhibit both IMPase and FBPase activities, and their crystal structures have been elucidated (4, 7, 23, 24).

The MJ0109 and AF2372 proteins were overexpressed in E. coli and purified (Fig. 2A and B). The purified MJ0109 and AF2372 each had a polypeptide mass of 28 kDa. This value was approximately the same as their individually calculated molecular masses (28.6 kDa for MJ0109 and 28.0 kDa for AF2372). In order to establish whether the MJ0109 and AF2372 proteins exhibit NADP(H)ase activities, their phosphatase activities toward various substrates were determined under the same assay conditions that were employed in the previous study for MJ0917 and compared with those of MJ0109 (4, 23), AF2372 (24), and MJ0917 (8) (Table 1). Some activities of MJ0917 were also determined in this study. MJ0109, AF2372, and MJ0917 exhibited high phosphatase activities toward NADP(H) and also toward the NADP(H) analogues containing 2'-phosphate (2'-AMP and 2'-phosphoadenosine 5'-phosphate [2'-PAP]) but not toward the NADP(H) analogues lacking 2'-phosphate (3'-AMP, 3'-PAP, and 5'-AMP) (Fig. 1). Obviously, these archaeal IMPase homologues showed high phosphatase activity toward the 2'-phosphate attached to the adenosine moiety. The kinetic values of the activities of MJ0109 and AF2372 toward NADP⁺ and NADPH were comparable to those toward FBP and IMP (Table 2). This implies that MJ0109 and AF2372 and probably the other archaeal IMPase homologues physiologically function as NADP(H)ase. The NADPase and NADPHase activities of MJ0109 and AF2372 were tolerant of LiCl in a manner similar to that of the IMPase and FBPase activities of the two enzymes (Table 3) (4, 24). In addition, we demonstrated that MJ0917 and particularly MJ0109 and AF2372 exhibited high IPPase, i.e., D-myo-inositol-1,4-bisphosphate 1-phosphatase, activity.

View larger version (37K): [in this window] [in a new window]

FIG. 2. SDS-PAGE results for (A) purified MJ0109, (B) purified AF2372, (C) E. coli cell extracts, and (D) purified CysQ. (A, B, and D) Lane 1, protein markers (Bio-Rad Laboratories, Hercules, CA); lane 2, the purified enzyme (MJ0109, 1.6 µg; AF2372, 2.6 µg; and CysQ, 1.8 µg). (C) Lane 1, protein markers. The other lanes indicate cell extracts (25 µg protein) of E. coli cells carrying pCA24N alone (lane 2), cysQ (lane 3), fbp (lane 4), and suhB (lane 5) in pCA24N.

View this table: [in this window] [in a new window]

TABLE 1. Phosphatase activities of several enzymes at 1.0 mM substrate concentrationa

View this table: [in this window] [in a new window]

TABLE 2. Kinetic constants for phosphatase activitiesa

View this table: [in this window] [in a new window]

TABLE 3. IC50 values of LiCl for phosphatase activitiesa

Collectively, we revealed that archaeal IMPases show broader substrate specificity values than those reported previously (3, 19, 23, 24). The IPPase activity of archaeal IMPase was considered to be compatible with the fact that archaeal IMPases belong to the IMPase/FBPase-like family (1, 26). Furthermore, the NADP(H)ase activities found in archaeal IMPase led us to hypothesize that some eukaryotic or eubacterial proteins belonging to this family might show NADP(H)ase activities. NADP(H)ase has not been identified in eubacteria and eukaryotes.

NADP(H)ase activity of the E. coli protein belonging to the IMPase/FBPase-like family.
To confirm this hypothesis, we chose the eubacterium E. coli that possesses SuhB (IMPase), Fbp (FBPase), and CysQ (the SuhB homologue) as the known proteins belonging to this family. There is no other primary structural homologue of SuhB or Fbp in E. coli. The CysQ protein is known to possess 3'-PAPase and IPPase activities (22); however, no other information on the properties of the CysQ protein is available.

As revealed by SDS-PAGE, SuhB, Fbp, and CysQ were overexpressed in E. coli cells in approximately the same amounts as the soluble proteins were (Fig. 2C). The cell extracts (0.58 µg protein) expressing each of the SuhB, Fbp, and CysQ proteins showed IMPase, FBPase, and 3'-PAPase activities. After 15-min reactions in the presence of IMP, FBP, and 3'-PAP, the extracts released 22.8 ± 0.3, 33.1 ± 0.1, and 41.5 ± 0.6 nmol P_i (means ± standard deviations for three determinations), respectively. On the other hand, the cell extracts (0.58 µg protein) from control cells carrying the vector alone produced merely 0, 0.37 ± 0.12, and 1.39 ± 0.10 nmol P_i from IMP, FBP, and 3'-PAP, respectively. As illustrated in Fig. 3, high NADP(H)ase activities were detected in the extract containing CysQ but not in those containing SuhB and Fbp.

View larger version (10K): [in this window] [in a new window]

FIG. 3. NADP(H)ase activities shown by (A) 5.8 µg protein and (B) 43.5 µg protein in each of the cell extracts derived from cells carrying suhB, fbp, and cysQ in pCA24N and pCA24N alone. The quantities of Pi released from NADP+ and NADPH after 15-min reactions were determined as described in Materials and Methods and are indicated by white (Pi from NADP+) and gray (Pi from NADPH) bars, respectively. Values are presented as the means ± standard deviations for three determinations.

Properties of CysQ.
In order to gain a further understanding of the kinetic properties of CysQ, we purified this enzyme. The purified CysQ had a polypeptide mass of 28 kDa (Fig. 2D), which is consistent with the calculated value (29.2 kDa). For maximum activities of 3'-PAPase and NADP(H)ase, CysQ required 0.20 mM and 10 mM MgCl₂ at pH 8.0, respectively. In the presence of 10 mM MgCl₂, CysQ showed maximum activities of 3'-PAPase and NADP(H)ase at pH 6.4 and pH 8.0, respectively. The 3'-PAPase activity was decreased to approximately 70% in the presence of 10 mM MgCl₂ and to approximately 50% at pH 8.0. In contrast, the NADP(H)ase activities were undetectable at pH 6.4 or in the presence of 0.2 mM MgCl₂. Hence, we determined the phosphatase activity of CysQ toward various substrates at pH 8.0 in the presence of 10 mM MgCl₂.

CysQ exhibited high 3'-PAPase and IPPase activities (as previously reported [22]); high 2'-PAP phosphatase (2'-PAPase); moderate NADP(H)ase, FBPase, and 3'-AMPase activities; and little or no phosphatase activities toward 2'-AMP and 5'-AMP (Table 1). The k_cat/K_m values for 2'-PAP and 3'-PAP were considerably higher than those for NADP⁺, NADPH, and FBP (Table 2), suggesting that CysQ is a 2'-PAPase or 3'-PAPase (Table 2). The IPPase activity of CysQ was previously shown to be kinetically less effective than the 3'-PAPase activity (22). Furthermore, the known phenotype of the cysQ mutant is slow growth in the absence of sulfite or cysteine under aerobic conditions (16). It has been proposed that the physiological role of 3'-PAPase is the removal of 3'-PAP that inhibits sulfotransferase, which is required for sulfate assimilation (20). From these results, we infer that CysQ functions physiologically as 3'-PAPase and that another "true" NADP(H)ase should exist in E. coli.

The 2'-PAPase and 3'-PAPase activities of CysQ were as sensitive to LiCl as were the 3'-PAPase activities of rice and yeast (Saccharomyces cerevisiae) (50% inhibitory concentration values of 0.20 and 0.10 mM, respectively) (15, 17). On the other hand, the NADPase, NADPHase, and FBPase activities of CysQ were as resistant to LiCl as the activities of the archaeal IMPase homologues were (Table 3).

Possibility that the IMPase homologue functions as physiological NADP(H)ase.
Although this study excluded the physiological role of CysQ as an NADP(H)ase in E. coli, genomic data in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.ad.jp/japanese/) still appear to support the possibility that the IMPase homologue functions as physiological NADP(H)ase in at least the other eubacteria. Cluster analysis using the KEGG database revealed that the IMPase homologue gene and the NUDIX protein gene form operon-like structures in 16 genomic sequences of some proteobacteria, including Vibrio, Pseudomonas, and Xanthomonas. The NUDIX protein has a domain that could hydrolyze the pyrophosphate moiety (11). These facts imply that NADP⁺ is initially dephosphorylated to NAD⁺ by the IMPase homologue and then degraded to NMN⁺ and 5'-AMP by the NUDIX protein in these proteobacteria (Fig. 1). Accordingly, in the archaeon M. kandleri, the IMPase homologue MK0741 is a fusion protein consisting of the IMPase homologue (E value of 2.1e-0.7) and the MazG nucleotide pyrophosphatase domain (E value of 4.3e-0.5). It is also possible that the IMPase homologue initially dephosphorylates NADP⁺ to NAD⁺, and the MazG nucleotide pyrophosphatase domain then degrades NAD⁺ to NMN⁺ and 5'-AMP (Fig. 1) in M. kandleri. MK0741 forms an operon-like structure with NAD kinase (MK0742) in M. kandleri (8).

 
We thank H. Mori of the Nara Institute of Science and Technology as well as the National BioResource Project (NIG, Japan) for providing us with the E. coli K-12 AG1 ASKA GFP-free strains.

 
* Corresponding author. Mailing address: Department of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. Phone: 81-774-38-3766. Fax: 81-774-38-3767. E-mail: kmurata{at}kais.kyoto-u.ac.jp

Published ahead of print on 6 July 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1
1. Andreeva, A., D. Howorth, S. E. Brenner, T. J. Hubbard, C. Chothia, and A. G. Murzin. 2004. SCOP database in 2004: refinements integrate structure and sequence family data. Nucleic Acids Res. 32:D226-D229.[Abstract/Free Full Text]
2
2. Berridge, M. E. 1993. Inositol triphosphate and calcium signalling. Nature 361:315-325.[CrossRef][Medline]
3
3. Chen, L., and M. F. Roberts. 1999. Characterization of a tetrameric inositol monophosphatase from the hyperthermophilic bacterium Thermotoga maritima. Appl. Environ. Microbiol. 65:4559-4567.[Abstract/Free Full Text]
4
4. Chen, L., and M. F. Roberts. 1998. Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme. Appl. Environ. Microbiol. 64:2609-2615.[Abstract/Free Full Text]
5
5. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756-1758.
6
6. Chifflet, S., A. Torriglia, R. Chiesa, and S. Tolosa. 1988. A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases. Anal. Biochem. 168:1-4.[CrossRef][Medline]
7
7. Johnson, K. A., L. Chen, H. Yang, M. F. Roberts, and B. Stec. 2001. Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities. Biochemistry 40:618-630.[CrossRef][Medline]
8
8. Kawai, S., C. Fukuda, T. Mukai, and K. Murata. 2005. MJ0917 in archaeon Methanococcus jannaschii is a novel NADP phosphatase/NAD kinase. J. Biol. Chem. 280:39200-39207.[Abstract/Free Full Text]
9
9. Kawai, S., S. Mori, T. Mukai, and K. Murata. 2004. Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: implication in regulation of NAD⁺/NADP⁺ balance. J. Basic Microbiol. 44:185-196.[CrossRef][Medline]
10
10. Kitagawa, M., T. Ara, M. Arifuzzaman, T. Ioka-Nakamichi, E. Inamoto, H. Toyonaga, and H. Mori. 2005. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 12:291-299.[Abstract/Free Full Text]
11
11. Koonin, E. V. 1993. A highly conserved sequence motif defining the family of MutT-related proteins from eubacteria, eukaryotes and viruses. Nucleic Acids Res. 21:4847.[Free Full Text]
12
12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
13
13. López-Coronado, J. M., J. M. Belles, F. Lesage, R. Serrano, and P. L. Rodriguez. 1999. A novel mammalian lithium-sensitive enzyme with a dual enzymatic activity, 3'-phosphoadenosine 5'-phosphate phosphatase and inositol-polyphosphate 1-phosphatase. J. Biol. Chem. 274:16034-16039.[Abstract/Free Full Text]
14
14. Lundquist, R., and B. M. Olivera. 1971. Pyridine nucleotide metabolism in Escherichia coli. I. Exponential growth. J. Biol. Chem. 246:1107-1116.[Abstract/Free Full Text]
15
15. Murguía, J. R., J. M. Belles, and R. Serrano. 1995. A salt-sensitive 3'(2'),5'-bisphosphate nucleotidase involved in sulfate activation. Science 267:232-234.[Abstract/Free Full Text]
16
16. Neuwald, A. F., B. R. Krishnan, I. Brikun, S. Kulakauskas, K. Suziedelis, T. Tomcsanyi, T. S. Leyh, and D. E. Berg. 1992. cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth. J. Bacteriol. 174:415-425.[Abstract/Free Full Text]
17
17. Peng, Z., and D. P. Verma. 1995. A rice HAL2-like gene encodes a Ca²⁺-sensitive 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase and complements yeast met22 and Escherichia coli cysQ mutations. J. Biol. Chem. 270:29105-29110.[Abstract/Free Full Text]
18
18. Pilkis, S. J., and T. H. Claus. 1991. Hepatic gluconeogenesis/glycolysis: regulation and structure/function relationships of substrate cycle enzymes. Annu. Rev. Nutr. 11:465-515.[CrossRef][Medline]
19
19. Sato, T., H. Imanaka, N. Rashid, T. Fukui, H. Atomi, and T. Imanaka. 2004. Genetic evidence identifying the true gluconeogenic fructose-1,6-bisphosphatase in Thermococcus kodakaraensis and other hyperthermophiles. J. Bacteriol. 186:5799-5807.[Abstract/Free Full Text]
20
20. Schwenn, J. D., F. A. Krone, and K. Husmann. 1988. Yeast PAPS reductase: properties and requirements of the purified enzyme. Arch. Microbiol. 150:313-319.[CrossRef][Medline]
21
21. Shieh, H. L., and H. L. Chiang. 1998. In vitro reconstitution of glucose-induced targeting of fructose-1,6-bisphosphatase into the vacuole in semi-intact yeast cells. J. Biol. Chem. 273:3381-3387.[Abstract/Free Full Text]
22
22. Spiegelberg, B. D., J. P. Xiong, J. J. Smith, R. F. Gu, and J. D. York. 1999. Cloning and characterization of a mammalian lithium-sensitive bisphosphate 3'-nucleotidase inhibited by inositol 1,4-bisphosphate. J. Biol. Chem. 274:13619-13628.[Abstract/Free Full Text]
23
23. Stec, B., H. Yang, K. A. Johnson, L. Chen, and M. F. Roberts. 2000. MJ0109 is an enzyme that is both an inositol monophosphatase and the "missing" archaeal fructose-1,6-bisphosphatase. Nat. Struct. Biol. 7:1046-1050.[CrossRef][Medline]
24
24. Stieglitz, K. A., K. A. Johnson, H. Yang, M. F. Roberts, B. A. Seaton, J. F. Head, and B. Stec. 2002. Crystal structure of a dual activity IMPase/FBPase (AF2372) from Archaeoglobus fulgidus. The story of a mobile loop. J. Biol. Chem. 277:22863-22874.[Abstract/Free Full Text]
25
25. Turner, W. L., J. C. Waller, B. Vanderbeld, and W. A. Snedden. 2004. Cloning and characterization of two NAD kinases from Arabidopsis. Identification of a calmodulin binding isoform. Plant Physiol. 135:1243-1255.[Abstract/Free Full Text]
26
26. York, J. D., J. W. Ponder, and P. W. Majerus. 1995. Definition of a metal-dependent/Li⁺-inhibited phosphomonoesterase protein family based upon a conserved three-dimensional core structure. Proc. Natl. Acad. Sci. USA 92:5149-5153.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, September 2007, p. 5447-5452, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.02703-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Shi, F., Li, Y., Li, Y., Wang, X. (2009). Molecular properties, functions, and potential applications of NAD kinases. Acta Biochim Biophys Sin 41: 352-361 [Abstract] [Full Text]  
• Brown, G., Singer, A., Lunin, V. V., Proudfoot, M., Skarina, T., Flick, R., Kochinyan, S., Sanishvili, R., Joachimiak, A., Edwards, A. M., Savchenko, A., Yakunin, A. F. (2009). Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli. J. Biol. Chem. 284: 3784-3792 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fukuda, C. Articles by Murata, K. Search for Related Content PubMed PubMed Citation Articles by Fukuda, C. Articles by Murata, K. Agricola Articles by Fukuda, C. Articles by Murata, K.