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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk-KPR) displayed reductase activity specific for 2-oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes. Tk-KPR activity decreased dramatically in the presence of CoA and KPR activity in cell-free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Coenzyme A (CoA) plays important roles in a wide range of metabolic pathways in all three domains of life (Genschel, 2004; Leonardi et al., 2005a; Spry et al., 2008). The mechanisms of CoA biosynthesis have been studied in detail in bacteria and eukaryotes (Leonardi et al., 2005a; Spry et al., 2008). In these organisms, CoA is synthesized from pantothenate via five reactions catalysed by pantothenate kinase (PanK), phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). In microorganisms and plants, pantothenate can be generated de novo from pyruvate via 2-oxoisovalerate, which is then converted to pantothenate by three reactions catalysed by ketopantoate hydroxymethyltransferase (KPHMT), ketopantoate reductase (KPR) and pantothenate synthetase (PS). Animals and some pathogenic bacteria, such as Haemophilus influenzae and Streptococcus pneumoniae, do not possess these enzymes (Gerdes et al., 2002) and thus rely on exogenous pantothenate for CoA synthesis.

The mechanisms of CoA biosynthesis in archaea are still not well understood (Genschel, 2004; Atomi et al., 2013). The PPCS/PPCDC fusion protein from Methanocaldococcus jannaschii (Kupke and Schwarz, 2006) and the PPAT from Pyrococcus abyssi (Armengaud et al., 2003; Nálezkova et al., 2005) have been biochemically examined. We have previously clarified that in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes, pantoate kinase (PoK) and phosphopantothenate synthetase (PPS), are responsible for the conversion of pantoate to 4′-phosphopantothenate, replacing the PS and PanK utilized in bacteria and eukaryotes (Yokooji et al., 2009). PoK and PPS homologues are present on almost all archaeal genomes, with exceptions limited to Nanoarchaeum equitans and members of the Thermoplasmatales. The presence of a PoK/PPS system has also been indicated in the methanogenic archaeon Methanospirillum hungatei (Katoh et al., 2012). In Thermoplasmatales, Ferroplasma acidarmanus and Picrophilus torridus harbour genes that display structural similarity with bacterial PanK. The PanK homologue from P. torridus has been shown to exhibit PanK activity (Takagi et al., 2010), whereas KPHMT, KPR and PS have not been identified.

The synthesis of one molecule of CoA from 2-oxoisovalerate requires one molecule each of β-alanine and cysteine, as well as five molecules of ATP and an NADPH. As this is a costly process, it can be expected that organisms harbour regulation mechanisms to prevent excess synthesis of CoA. In Escherichia coli, PanK (type I PanK encoded by coaA), is dramatically inhibited by CoA, and is a key regulator of the pathway (Vallari et al., 1987; Rock et al., 2003). CoA inhibits PanK activity by competitive feedback inhibition with one of its substrates, ATP (Song and Jackowski, 1994; Yun et al., 2000). Acetyl-CoA, succinyl-CoA and other acyl-CoA derivatives also inhibit PanK from E. coli, although the effects are not as strong as that of CoA (Vallari et al., 1987). PPAT is another target of feedback inhibition by CoA in E. coli, but the effects are moderate compared with that of PanK (Miller et al., 2007). KPHMT is also inhibited by CoA; however, the concentration of CoA required is relatively high (> 1 mM) (Powers and Snell, 1976). These observations suggest that PanK is the key regulatory enzyme of CoA biosynthesis in E. coli.

The first eukaryotic PanK (type II PanK) was identified from Aspergillus nidulans. The enzyme is strongly inhibited by acetyl-CoA, and to a lower extent by CoA and malonyl-CoA, in a competitive manner with ATP (Calder et al., 1999). In plants, PanK from spinach is inhibited by malonyl-CoA, but not by CoA or acetyl-CoA (Falk and Guerra, 1993). In mice, multiple isoforms of PanK have been identified, among which two (PanK1α and PanK1β) are produced by the Pank1 gene (Rock et al., 2002). PanK1α is inhibited by CoA, acetyl- and malonyl-CoA, whereas PanK1β is only weakly inhibited by acetyl- and malonyl-CoA (Rock et al., 2002; Zhang et al., 2005). PanK2 is also inhibited by acetyl-CoA (Leonardi et al., 2007), while PanK3, which shows high similarity with PanK1β, is inhibited by CoA, acetyl-, malonyl- and palmitoyl-CoA (Zhang et al., 2005). The data imply that in eukaryotes also, CoA biosynthesis is regulated by feedback regulation on PanK.

In contrast to these two types of PanK, some bacteria, including Bacillus subtilis and Helicobacter pylori, harbour type III PanK enzymes encoded by coaX (Osterman and Overbeek, 2003; Brand and Strauss, 2005), which are not inhibited by CoA and acetyl-CoA. Another exceptional case is the PanK from Staphylococcus aureus, which although related to Type II PanK in sequence, is not inhibited by CoA. The intracellular concentration of CoA in S. aureus is considered to be relatively high, protecting the cell from oxidative stress (Leonardi et al., 2005b).

As most archaea utilize the PoK/PPS system and do not harbour a PanK (Yokooji et al., 2009), the regulation mechanisms of CoA biosynthesis in archaea are of interest. Contrary to our expectations, we recently found that both PoK and PPS from T. kodakarensis are not inhibited by CoA and acetyl-CoA, suggesting they are not targets of feedback inhibition (Ishibashi et al., 2012; Tomita et al., 2012). Similarly, the PoK and PPS from M. hungatei also are not inhibited by CoA and its derivatives (Katoh et al., 2012). Furthermore, the PanK from P. torridus, which is an exceptional archaeon that does not rely on the PoK/PPS system, is also not inhibited by CoA-related compounds (Takagi et al., 2010). The regulatory mechanisms of CoA biosynthesis in archaea thus seem to differ from those of the bacteria and eukaryotes.

Here, we describe the identification and first characterization of an archaeal ketopantoate reductase (KPR), encoded by TK1968 in T. kodakarensis. The enzyme was NAD-dependent, distinct to the NADP-dependent enzymes from bacteria/eukaryotes, and strongly inhibited by CoA. In addition, we found that KPHMT activity was not affected by CoA, demonstrating that KPR is the primary target of feedback inhibition for the regulation of CoA biosynthesis in this archaeon.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

A candidate for the gene encoding archaeal ketopantoate reductase

Among the genes in T. kodakarensis, TK1968 was the only gene that encoded a protein with similarity to the KPR proteins from E. coli (Wilken et al., 1975; Elischewski et al., 1999) and Saccharomyces cerevisiae (King and Wilken, 1972; King et al., 1974; Wilken et al., 1975), with identities of 29% and 20% respectively (Fig. 1). The KPR from E. coli has been characterized in detail in terms of its substrate specificity (Zheng and Blanchard, 2003), kinetic and mechanistic properties (Zheng and Blanchard, 2000a,b; 2003; Ciulli et al., 2007) and its structure (Matak-Vinković et al., 2001; Lobley et al., 2005; Ciulli et al., 2007). The TK1968 protein harbours a predicted Rossman-fold domain containing a GXGXXG motif (Kleiger and Eisenberg, 2002) at its N-terminus, as in the cases of KPR proteins from E. coli and S. cerevisiae. The motif is common to nucleotide-binding domains, and the motif in E. coli KPR is involved in NADP+ binding (Lobley et al., 2005). Many other residues that have been reported to be important for catalysis in the KPR from E. coli (Matak-Vinković et al., 2001; Lobley et al., 2005; Ciulli et al., 2007) are also conserved in the TK1968 protein. We thus predicted that the TK1968 gene encodes the archaeal ketopantoate reductase in T. kodakarensis, and selected the gene for further analyses.

figure

Figure 1. A sequence alignment of the TK1968 protein and KPR enzymes from Escherichia coli and Saccharomyces cerevisiae. Ec, Tk and Sc represent KPR from E. coli (ABG68514), the TK1968 protein (BAD86157) and KPR from S. cerevisiae (AAB68390) respectively. The GXGXXG motif is indicated in a grey box. Amino acid residues that are involved in NADP+ or pantoate binding in E. coli KPR (Ciulli et al., 2007) are indicated with circles or triangles respectively. Sequences were aligned with the clustalw program provided by the DNA Data Bank of Japan (DDBJ).

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Gene disruption of TK1968

To examine the contribution of TK1968 to CoA and pantoate biosynthesis in T. kodakarensis, we constructed a gene disruption strain and compared its phenotype with that of the host strain, T. kodakarensis KUW1. The disruption plasmid was designed so that gene disruption would occur via single-crossover insertion of the plasmid, followed by pop-out recombination (Fig. S1A). Cells that had undergone single-crossover insertion were enriched by growing the transformants in a uracil-free medium, and cells that had further undergone pop-out recombination were selected on solid media that included 5-FOA and CoA (1 mM). CoA was added due to the possibility that the transformant could not grow without CoA, as was observed in the disruption strains of pantoate kinase (pok) and phosphopantothenate synthetase (pps) genes (Yokooji et al., 2009). PCR analysis (Fig. S1B) and DNA sequencing of genomic DNA confirmed the isolation of a transformant deleted of the TK1968 gene.

Growth characteristics of the gene disruption strain of TK1968

The TK1968 gene disruption strain (ΔTK1968) and its host strain KUW1 were grown in ASW-YT-S0 medium (Fig. 2). As a result, ΔTK1968 displayed a slower growth rate and a lower cell yield compared with its host strain, KUW1. When we added 1 mM pantoate to the medium, the product of the KPR reaction, the growth defects observed in the ΔTK1968 strain were complemented, with both growth rate and cell yield fully recovered. This indicates that in the ΔTK1968 strain, pantoate is the rate-limiting substrate for growth of this strain, and that the TK1968 gene is necessary to supply sufficient levels of pantoate in T. kodakarensis.

figure

Figure 2. Growth characteristics of T. kodakarensis KUW1 and ΔTK1968. Cells were cultivated in ASW-YT-S0 medium at 85°C. Symbols: circles, KUW1; squares, ΔTK1968; and triangles, ΔTK1968 grown in medium supplemented with 1 mM pantoate.

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Production and purification of the recombinant TK1968 protein

The TK1968 gene was expressed in E. coli, and a soluble protein was obtained. The protein was purified by heat treatment, followed by anion-exchange chromatography and gel filtration chromatography. A single band corresponding to the molecular mass of the recombinant TK1968 protein, calculated as 34 048 Da from its primary sequence, was observed after SDS-PAGE (Fig. S2). The purified protein was examined for KPR activity, and applied to gel filtration chromatography to determine its quaternary structure. The majority of the protein (∼ 90%) eluted at a volume corresponding to a molecular mass of ∼ 60 kDa, indicating that the protein assembles as a dimer. A minor fraction of the protein eluted at a volume corresponding to a tetramer (data not shown).

Basic enzymatic properties of the TK1968 protein

We first examined KPR activity for the recombinant TK1968 protein with NADPH or NADH at 85°C. The recombinant protein displayed KPR activity, and higher activity was observed when NADH was used as an electron donor (data not shown). As the degree of thermal degradation of NAD(P)H at 85°C was high and hampered our activity measurements, further experiments were carried out with NADH at 70°C.

We examined the effect of pH on the KPR reaction, and observed high activity at neutral pH with a maximum at pH 6.4 in 50 mM MES-NaOH buffer (Fig. 3A). In terms of temperature, TK1968 protein showed highest activity at 90°C (Fig. 3B). An Arrhenius plot of the data showed linearity between 30°C and 65°C, and 65°C and 85°C (Fig. 3C). The activation energies of the reaction were calculated to be 90.9 kJ mol−1 (30–65°C) and 65.4 kJ mol−1 (65–85°C). Thermostability of the TK1968 protein was examined by incubating the protein at 60°C, 70°C, 80°C and 90°C for various periods of time and by measuring residual activity (Fig. 3D). No decrease in KPR activity was detected at all temperatures for at least 24 h, indicating that the TK1968 protein from T. kodakarensis is extremely thermostable.

figure

Figure 3. Basic enzymatic properties of Tk-KPR.

A. The effects of pH on KPR activity. Symbols: circles, MES (pH 5.2–7.1); squares, PIPES (pH 7.0–7.9); and diamonds, HEPES (pH 7.2–8.2).

B. The effects of temperature on KPR activity.

C. An Arrhenius plot of the data shown in (B).

D. Thermostability of Tk-KPR. Tk-KPR was incubated at various temperatures for the indicated periods of time, and residual activity was measured. Symbols: circles, 60°C; squares, 70°C; diamonds, 80°C; and triangles, 90°C.

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Substrate specificity for 2-oxo acids

We examined the substrate specificity of the recombinant TK1968 protein towards various 2-oxo acids; 2-oxopantoate, pyruvate, oxaloacetate and 2-oxoglutarate. KPR reactions were carried out in the presence of 1 or 10 mM of 2-oxo acid. As a result, the TK1968 protein recognized only 2-oxopantoate, providing further evidence that TK1968 encodes the KPR of T. kodakarensis (data not shown). The protein was thus designated Tk-KPR.

Kinetic examinations of the ketopantoate reductase reaction

Kinetic studies on the forward and reverse KPR reactions were performed by varying the concentration of one substrate in the presence of a constant concentration (0.2 mM in the forward reaction and 1 mM in the reverse reaction) of the other. The kinetics towards all substrates [2-oxopantoate, NADH and NADPH (forward reaction), d-pantoate, l-pantoate and NAD+ (reverse reaction)] followed Michaelis–Menten kinetics (Fig. 4A–E). The obtained kinetic parameters are shown in Table 1. Although the activity with NADP+ in the reverse reaction was detected, the initial velocities were too low to perform a kinetic analysis. First of all, the results indicate that Tk-KPR prefers NADH rather than NADPH, which is distinct to the bacterial and eukaryotic KPR enzymes that prefer NADPH (King and Wilken, 1972; Shimizu et al., 1988; Zheng and Blanchard, 2003). Second, from a kinetic point of view, the forward reaction (e.g. kcat/Km = 3.83 s−1 μM−1) is favoured over the reverse reaction (e.g. kcat/Km = 0.029 s−1 μM−1). A third point is that d-pantoate was much more preferred over l-pantoate in the reverse reaction, suggesting that the enzyme generates d-pantoate from 2-oxopantoate in the forward reaction. We have previously reported a kinetic analysis of PoK, which catalyses the phosphorylation of pantoate, using d-pantoate as the substrate (Tomita et al., 2012). In order to confirm whether PoK also favours d-pantoate, we examined PoK activity towards l-pantoate. PoK preferred d-pantoate as a substrate, with over 100-fold higher activity towards d-pantoate compared with l-pantoate at a concentration of 1 mM (data not shown).

figure

Figure 4. Kinetic examination of Tk-KPR.

A. Initial velocities of the forward reaction (2-oxopantoate reduction) with varying concentrations of 2-oxopantoate and 0.2 mM NADH.

B. Initial velocities of the forward reaction with varying concentrations of NAD(P)H and 0.2 mM 2-oxopantoate. Symbols: circles, NADH; squares, NADPH.

C. Initial velocities of the reverse reaction (pantoate oxidation) with varying concentrations of d-pantoate and 1 mM NAD+.

D. Initial velocities of the reverse reaction with varying concentrations of l-pantoate and 1 mM NAD+.

E. Initial velocities of the reverse reaction with varying concentrations of NAD+ and 1 mM d-pantoate.

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Table 1. Kinetic parameters of ketopantoate reductase
DirectionForward (reduction of 2-oxopantoate)Reverse (oxidation of pantoate)
Substrate2-OxopantoateNADHNADPHd-Pantoatel-PantoateNAD+
  1. KPR activity with varying concentrations of 2-oxopantoate was measured in the presence of 0.2 mM NADH.

  2. KPR activity with varying concentrations of NADH was measured in the presence of 0.2 mM 2-oxopantoate.

  3. KPR activity with varying concentrations of d/l-pantoate was measured in the presence of 1 mM NAD+.

  4. KPR activity with varying concentrations of NAD+ was measured in the presence of 1 mM d/l-pantoate.

Vmax (μmol min−1 mg−1)40.7 ± 1.134.7 ± 0.43.92 ± 0.046.70 ± 0.165.60 ± 0.244.26 ± 0.12
Km (μM)6.03 ± 0.753.01 ± 0.201.35 ± 0.11130 ± 102040 ± 22040.9 ± 3.1
kcat (s−1)23.1 ± 0.619.7 ± 0.22.22 ± 0.023.80 ± 0.093.17 ± 0.142.41 ± 0.07
kcat/Km (s−1 μM−1)3.836.541.640.0290.0020.06

Effect of CoA and acetyl-CoA on Tk-KPR activity

As we have previously observed that PoK and PPS activities are not inhibited by CoA or acetyl-CoA (Ishibashi et al., 2012; Tomita et al., 2012), the enzyme that acts as the target of feedback inhibition, if present at all, has not been identified in the Archaea. We thus examined whether Tk-KPR is regulated by the presence of CoA or acetyl-CoA. KPR reactions were carried out in the presence of various concentrations of CoA or acetyl-CoA. As a result, we detected a dramatic decrease in activity with the addition of CoA or acetyl-CoA (Fig. 5A), indicating that the KPR activity is controlled by feedback inhibition by CoA. In the presence of 0.2 mM 2-oxopantoate and 0.2 mM NADH, the IC50 values of CoA and acetyl-CoA were 17 μM and 40 μM respectively. In order to clarify the mechanism of this inhibition, we carried out kinetic analysis towards NADH in the presence of 0.2 mM 2-oxopantoate and various concentrations of CoA (Fig. 5B). As a result, the concentration of CoA affected the Km values whereas the Vmax values were relatively constant (Table 2), suggesting that CoA inhibits Tk-KPR by competing with NADH. Using the data of kinetic analyses in the presence of 0, 0.2, 0.4 or 0.6 μM CoA shown in Fig. 5B, we calculated the Ki value of CoA based on the equation, v = Vmax[NADH]/{(1 + [CoA]/Ki)Km + [NADH]}, where v is the initial velocity, [NADH] and [CoA] are the concentrations of NADH and CoA, respectively, Km is the Michaelis constant towards NADH, and Vmax is the maximum velocity (Table 2). The Ki value of CoA towards the Tk-KPR reaction was 42 ± 3 nM, indicating a high sensitivity of the enzyme towards accumulation of CoA.

figure

Figure 5. Effects of CoA and acetyl-CoA on Tk-KPR activity.

A. Activity measurements with varying concentrations of CoA (circles) or acetyl-CoA (squares), and 0.2 mM 2-oxopantoate and 0.2 mM NADH.

B. Kinetic examinations of KPR reaction with varying concentrations of NADH and CoA, and 0.2 mM 2-oxopantoate. Symbols: closed circles, without CoA; open circles, 0.2 μM CoA; closed squares, 0.4 μM CoA; and open squares, 0.6 μM CoA.

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Table 2. Kinetic parameters of Tk-KPR towards NADH in the presence of different concentrations of CoA
 Vmax (μmol min−1 mg−1)Km (μM)Ki (μM)
  1. a

    Kinetic parameters were calculated using the data obtained in the presence of the indicated CoA concentrations with the Michaelis–Menten equation.

  2. b

    Kinetic parameters were calculated using the data of kinetic analyses in the presence of 0, 0.2, 0.4 or 0.6 μM CoA shown in Fig. 5B with the equation v = Vmax[NADH]/{(1 + [CoA]/Ki)Km + [NADH]}.

  3. KPR activity with varying concentrations of NADH was measured in the presence of 0.2 mM 2-oxopantoate and different concentrations of CoA.

No CoAa41.7 ± 0.43.6 ± 0.2
0.2 μM CoAa40.6 ± 0.915.3 ± 1.3
0.4 μM CoAa39.8 ± 0.836.6 ± 2.1
0.6 μM CoAa40.5 ± 1.552.2 ± 4.9
0–0.6 μM CoAb41.5 ± 0.43.5 ± 0.20.042 ± 0.003

Examination of KPR activity in the cell-free extracts of T. kodakarensis

We next measured KPR activity in the cell-free extracts of T. kodakarensis grown in the presence or absence of CoA (Fig. 6). An NADH-dependent KPR activity could clearly be observed in KUW1 cells grown in the absence of CoA. In the ΔTK1968 cells however, no such activity could be detected. NADPH-dependent activity was also not present, confirming that TK1968 is the gene primarily responsible for KPR activity in T. kodakarensis. Addition of CoA to the medium did not have any significant effect on KPR activity levels. This is a similar result to the KPR from Pseudomonas maltophilia, whose activity levels were not affected by the addition of CoA to the medium (Shimizu et al., 1988). Addition of CoA to the assay reaction mixture resulted in a large decrease in KPR activity, consistent with our results using the purified, recombinant protein.

figure

Figure 6. KPR activity in cell-free extracts of strains KUW1 and ΔTK1968. Strains were grown in ASW-YT-Pyr medium in the presence (CoA+) or absence (CoA) of 1 mM CoA. KPR activity was measured with 0.2 mM 2-oxopantoate and 0.2 mM NADH, in the presence (grey bar) or absence (white bar) of 1 mM CoA in the reaction mixture. All measurements were carried out in triplicate. ND, not detected.

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Production and purification of the recombinant TK0363 protein

Our results suggest that CoA biosynthesis in T. kodakarensis can be regulated by feedback inhibition on KPR. We have previously reported that activities of PoK and PPS are not affected by the presence of CoA/acetyl-CoA (Ishibashi et al., 2012; Tomita et al., 2012; Atomi et al., 2013). In order to gain further support that regulation is mainly carried out at the KPR reaction, we examined the enzymatic properties of KPHMT, which catalyses the first step of the CoA biosynthesis pathway. TK0363 is the only gene that encodes a protein with similarity to the KPHMT proteins from E. coli (Powers and Snell, 1976; Teller et al., 1976; Jones et al., 1993), Mycobacterium tuberculosis (Sugantino et al., 2003) and A. nidulans (Kurtov et al., 1999), with identities of 28%, 23% and 19% respectively. The crystal structures of the KPHMT from E. coli and M. tuberculosis have been elucidated (Chaudhuri et al., 2003; Schmitzberger et al., 2003; von Delft et al., 2003), and residues proposed to be involved in 2-oxoisovalerate-binding (Ser46, Lys112, His136 and Glu181) and Mg2+-binding (Asp45, Asp84 and Glu114) in the KPHMT from E. coli (von Delft et al., 2003) are conserved in the TK0363 protein.

The TK0363 gene was expressed in E. coli, and a soluble protein was obtained. The protein was purified using methods identical to those for Tk-KPR. A single band corresponding to the molecular mass of the recombinant TK0363 protein, calculated as 31 742 Da from its primary sequence, was observed after SDS-PAGE (Fig. S2).

Examination of KPHMT activity of the TK0363 protein and effects of CoA

We first examined KPHMT activity for the recombinant TK0363 protein (Tk-KPHMT) with 2-oxoisovalerate and N5, N10-methylenetetrahydrofolate or formaldehyde at 85°C. The recombinant protein displayed KPHMT activity with both N5, N10-methylenetetrahydrofolate and formaldehyde. In the presence of 50 mM 2-oxoisovalerate and 2 mM N5, N10-methylenetetrahydrofolate, the reaction rate was 1.31 μmol min−1 mg−1. When N5, N10-methylenetetrahydrofolate was replaced with formaldehyde, we observed activity levels of 1.06 μmol min−1 mg−1, suggesting that although utilized, tetrahydrofolate is not essential for the Tk-KPHMT reaction. As N5, N10-methylenetetrahydrofolate displays low solubility, we carried out a kinetic analysis of the Tk-KPHMT reaction in the presence of 2 mM N5, N10-methylenetetrahydrofolate and varying concentrations of 2-oxoisovalerate. The Km value for 2-oxoisovalerate was 4.73 ± 0.72 mM and the kcat value was 0.76 ± 0.02 s−1. We were also able to examine the kinetics of the reaction with 50 mM 2-oxoisovalerate and varying concentrations of formaldehyde, resulting in a Km value of 4.92 ± 0.12 mM for formaldehyde and a kcat of 1.97 ± 0.01 s−1. The higher kcat in the latter reaction is due to the high concentrations of formaldehyde in the reaction compared with N5, N10-methylenetetrahydrofolate.

We next examined whether the activity of Tk-KPHMT is affected by the presence of CoA. KPHMT reactions with 5 mM 2-oxoisovalerate and 2 mM N5, N10-methylenetetrahydrofolate were carried out in the presence of 0, 0.1, 0.5 and 1 mM of CoA. As a result, differences in KPHMT activity due to the presence of CoA were less than 6% of activity in the absence of CoA. The results indicate that Tk-KPHMT is neither activated nor inhibited by CoA.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we have performed the first detailed biochemical and genetic analyses of an archaeal KPR that catalyses the reduction of 2-oxopantoate to pantoate, the substrate of the following PoK reaction in CoA biosynthesis. We have also performed an initial characterization of an archaeal KPHMT, focusing on the effects of CoA on the reaction. Our results demonstrate that TK1968 encodes the KPR in T. kodakarensis. Disruption of TK1968 leads to a condition in which pantoate is the rate-limiting substrate for growth, and KPR activity can no longer be detected in T. kodakarensis cell-free extracts. In addition, the TK1968 protein displayed KPR activity with a strict specificity towards 2-oxopantoate. Although with lower growth rates and cell yields, the ΔTK1968 strain could still grow in ASW-YT-S0 medium without the addition of CoA and its precursors. We have previously observed that the PoK and PPS gene disruption strains could not grow at all under these conditions (Yokooji et al., 2009), indicating that metabolites downstream of 4-phosphopantoate cannot be supplied by medium components. This suggests that the retarded growth of the ΔTK1968 strain is due to either a minor, alternative route that can generate pantoate, or a small amount of pantoate present in the ASW-YT-S0 medium. In order to examine these possibilities, the ΔTK1968 strain was grown in ASW-AA-S0 medium, a completely synthetic medium which does not contain intermediates of the CoA biosynthesis pathway, such as pantoate. As a result, although retarded, we were still able to observe cell growth (data not shown), indicating that another enzyme(s) is capable of providing low levels of pantoate. There are a number of putative genes on the T. kodakarensis genome whose products are predicted to catalyse the reduction of 2-oxocarboxylic acids, including malic enzyme, two lactate dehydrogenases, 3-phosphoglycerate dehydrogenase and 3-isopropylmalate dehydrogenase. One of these may be responsible for providing minimal amounts of pantoate in the ΔTK1968 strain.

In addition, we demonstrated that TK0363 encodes a protein with KPHMT activity. Tk-KPHMT utilized not only N5, N10-methylenetetrahydrofolate but also free formaldehyde, with comparable reaction rates. This is similar to the KPHMT from M. tuberculosis. This enzyme can catalyse the KPHMT reaction in the absence of N5, N10-methylenetetrahydrofolate, with a turnover number of 135 min−1 observed with 50 mM 2-oxoisovalerate and 50 mM free formaldehyde, higher than the kcat value calculated with N5, N10-methylenetetrahydrofolate (47 min−1) (Sugantino et al., 2003).

Tk-KPR displayed several interesting features, including a preference for NADH rather than NADPH as an electron donor, distinct to the KPR from bacteria and eukaryotes (King and Wilken, 1972; Wilken et al., 1975; Shimizu et al., 1988; Zheng and Blanchard, 2003). Moreover, the enzyme was inhibited by CoA, suggesting that the enzyme is the target of feedback inhibition in regulating CoA biosynthesis in the archaeon. We have previously reported that PoK and PPS, enzymes specific for archaea, from T. kodakarensis are not affected by CoA and acetyl-CoA (Ishibashi et al., 2012; Tomita et al., 2012; Atomi et al., 2013). The behaviour of Tk-KPR thus solves the apparent lack of a regulation system in archaea corresponding to those in bacteria and eukaryotes relying on feedback inhibition of PanK (Fig. 7). In addition, we found that Tk-KPHMT activity is not affected by CoA. This indicates that, among the first four enzymes in archaeal CoA biosynthesis (KPHMT, KPR, PoK, PPS), only KPR is inhibited by CoA in T. kodakarensis, strongly suggesting that KPR is the primary target of feedback inhibition by CoA in this organism. The KPR-independent route for pantoate synthesis, suggested by our genetic studies, may not be regulated at all, but the clear defects in growth observed in the ΔTK1968 imply that the main route for pantoate biosynthesis in T. kodakarensis is dependent on Tk-KPR.

figure

Figure 7. An illustration of the regulation mechanisms of CoA biosynthesis in archaea, bacteria and eukaryotes. In bacteria and eukaryotes, PanK is the target of feedback inhibition by CoA. In archaea, the PoK/PPS system functions in place of the PS/PanK system. PoK and PPS have been shown to be unaffected by CoA (Ishibashi et al., 2012; Tomita et al., 2012). This study reveals that KPR is inhibited by CoA, and KPHMT is not.

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Although previous studies have not directly examined whether KPR enzymes from E. coli and S. cerevisiae are affected by CoA, PanK is considered the primary target in the regulation of CoA biosynthesis in both bacteria and eukaryotes (Jackowski and Rock, 1981; Robishaw et al., 1982). In E. coli, it has been reported that increases in the addition of β-alanine lead to increases in CoA content. CoA levels saturated with the addition of 8 μM β-alanine, but further increases led to excretion of pantothenate into the medium. The results clearly demonstrate that the supply of pantoate is not a limiting factor, and that PanK is the primary target for regulation of CoA biosynthesis (Jackowski and Rock, 1981). The archaeal regulation system targeting the KPR reaction seems favourable in terms of energy consumption compared with those at the PanK reaction utilized by bacteria and eukaryotes, as excess ATP-dependent conversion of pantoate (PS reaction in bacteria and eukaryotes, PoK in archaea) can be prevented. Regulation at PanK in mammalian cells is reasonable, as in organisms that rely on exogenous pantothenate, the PanK reaction corresponds to the first step in CoA biosynthesis.

In E. coli, CoA binds with PanK in a competitive manner towards one of its substrates, ATP. Structural studies have indicated that the phosphodiester binding sites of CoA and ATP overlap to one another (Song and Jackowski, 1994; Yun et al., 2000). In archaeal KPR, the results of our kinetic experiments shown in Fig. 5B suggest that CoA inhibits Tk-KPR in a competitive manner with NADH. As CoA and NADH share an ADP moiety, there is a possibility that the binding sites of CoA and NADH may overlap. Structural studies on Tk-KPR bound with its substrate or CoA will be necessary to elucidate the mechanisms governing the inhibition by CoA.

Clear homologues of the TK1968 gene are relatively widespread among the archaea. They are found in many of the genomes of Desulfurococcales, Thermoproteales, Archaeaoglobales, Halobacteriales, Methanocellales, Methanomicrobiales and Thermococcales. Tk-KPR homologues are also found in a limited number of species within the Methanococcales and Methanosarcinales. In contrast, they cannot be found in the Sulfolobales, Methanopyrales, Thermoplasmatales, Korarchaeota, Thaumarchaeota and Nanoarchaeota. In the organisms that harbour Tk-KPR homologues, the mechanisms of pantoate biosynthesis and regulation of CoA biosynthesis may be similar to those in T. kodakarensis. When we focus on the methanogens, besides the absence of Tk-KPR homologues, all organisms lack a gene homologous to Tk-KPHMT. This is most likely due to the fact that methanogens are considered to use methylenetetrahydromethanopterin (Keltjens et al., 1983), and not N5, N10-methylenetetrahydrofolate, as the hydroxymethyl group donor (Thauer, 1998). The route to pantoate biosynthesis (KPHMT and KPR) may have evolved in a different manner in the methanogens. In some bacteria, including Salmonella typhimurium, E. coli and Corynebacterium glutamicum, the ilvC gene, encoding acetohydroxyacid isomeroreductase which is involved in the biosynthesis of the branched-chained amino acids, has also been shown to catalyse the reduction of 2-oxopantoate (Primerano and Burns, 1983; Elischewski et al., 1999; Merkamm et al., 2003). Although archaeal ilvC gene has not been examined, there is a possibility that archaea that do not harbour a Tk-KPR homologue but do harbour an ilvC homologue utilize the latter for reduction of 2-oxopantoate. Indeed, all members of the Sulfolobales, Methanomicrobiales, Methanopyrales and Methanosarcinales harbour an ilvC homologue. This is also the case for Korarchaeota, Thaumarchaeota, two species of Thermoplasmatales (F. acidarmanus and P. torridus) and four species of Methanococcales. As most of these species do not possess a Tk-KPR homologue, this distribution raises the possibility that ilvC homologues may be responsible for the supply of pantoate in these organisms. An ilvC homologue is not present on the T. kodakarensis genome. In addition, a very recent study demonstrated that some pathogenic bacteria such as Francisella tularensis use a novel type KPR encoded by panG (Miller et al., 2012). However, its homologues are not conserved in any of the archaeal genomes.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains, media and culture conditions

Cultivation of T. kodakarensis KOD1 (Morikawa et al., 1994; Atomi et al., 2004; Fukui et al., 2005) and its derivative strains was performed under anaerobic conditions at 85°C in a nutrient-rich medium (ASW-YT) or a synthetic medium (ASW-AA). ASW-YT medium consists of 0.8× artificial seawater (ASW), 5.0 g l−1 yeast extract, 5.0 g l−1 tryptone and 0.8 mg l−1 resazurine. Prior to inoculation, 2.0 g l−1 elemental sulphur (ASW-YT-S0 medium) or 5.0 g l−1 sodium pyruvate (ASW-YT-Pyr medium) and Na2S were added to the medium until it became colourless. ASW-AA medium consists of 0.8× ASW, a mixture of 20 amino acids, modified Wolfe's trace minerals, a vitamin mixture and 2.0 g l−1 elemental sulphur (ASW-AA-S0 medium) (Robb and Place, 1995; Sato et al., 2003). In the case of plate culture used to isolate transformants, elemental sulphur and Na2S 9H2O were replaced with 2 ml of a polysulphide solution (10 g Na2S 9H2O and 3 g sulphur flowers in 15 ml H2O) per litre and 10 g l−1 Gelrite was added to solidify the medium. In the case of growth measurements, cells were first cultivated in ASW-YT-S0 medium supplemented with 1 mM CoA. Cells from this culture were next inoculated in ASW-YT-S0 medium without any supplement. Cells from this second culture were inoculated in various media to examine growth. Specific modifications of the medium to select and examine the auxotrophy of mutant strains are described in the respective sections. E. coli strains DH-5α and BL21-CodonPlus(DE3)-RIL, used for plasmid construction and heterologous gene expression, respectively, were cultivated at 37°C in Luria–Bertani (LB) medium containing ampicillin (100 mg l−1). Unless mentioned otherwise, all chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).

Construction of the plasmid for gene disruption of TK1968

The gene disruption plasmid for TK1968 was constructed by first amplifying the gene along with 1 kbp of its 5′- and 3′-flanking regions using the primer set 1968F1/1968R1 (5′-AACGTATGGCTCTGGCCTCTTCTTC-3′/5′-GCTATCTTCCTCGCGACGGCGAAG-3′). The fragment was inserted into the HincII restriction enzyme site of pUD3, which contains the pyrF marker gene cassette inserted in the ApaI site of pUC118. Inverse PCR was performed with 1968F2/1968R2 (5′-ATGTCACTGTTCGGTGGAAAAGAG-3′/5′-TCTATCACCTCGCGAGCTTCCTAAG-3′) to remove the coding region, and the amplified fragment was self-ligated. Sequences of the 5′- and 3′-flanking regions were confirmed.

Gene disruption of TK1968

Thermococcus kodakarensis strain KUW1 (ΔpyrF, ΔtrpE) (Sato et al., 2005), which shows uracil and tryptophan auxotrophy, was used as the host strain for TK1968 gene disruption. KUW1 was cultivated in ASW-YT-S0 medium for 12 h at 85°C. Cells were harvested, resuspended in 200 μl of 0.8× ASW, and incubated on ice for 30 min. After adding 3.0 μg of the gene disruption plasmid and further incubation on ice for an hour, cells were cultivated in ASW-AA-S0 medium without uracil for 24 h at 85°C. Cells were then harvested, diluted with 0.8× ASW, and spread onto solid ASW-YT-S0 medium supplemented with 10 g l−1 of 5-fluoroorotic acid (5-FOA), 60 mM NaOH and 1 mM CoA. Only cells that have undergone a pop-out recombination that removes the pyrF gene can grow in the presence of 5-FOA. After cultivation for 2 days at 85°C, transformants displaying 5-FOA resistance were isolated and cultivated in ASW-YT-S0 medium with 1 mM CoA. After cultivation, the genotypes of the isolated transformants were analysed by PCR with the primer sets 1968F3/1968R3 (5′-AACTGTGAAGTCGAAGAGTTCAAG-3′/5′-AGGATGCCTCGGATCAGGTCATGC-3′) and 1968F4/1968R4 (5′-GCAGTGCATAGGGAGGAACACGTGG-3′/5′-ATCCCCTCGAGATGAGGATCGTCCT-3′). Transformants whose amplified DNA products displayed the expected size were chosen, and relevant sequences were confirmed to have no unintended mutations.

Overexpression and purification of the recombinant TK1968 and TK0363 proteins

The TK1968 and TK0363 genes were overexpressed in E. coli. The coding regions of the TK1968 and TK0363 genes were amplified from the genomic DNA of T. kodakarensis KOD1 by PCR using the primer sets 1968F5/1968R5 (5′-GGGGAAAACATATGAGGATATACGTTCTCGGTGC-3′/5′-AAAAGAATTCTCAACACCCCTCGCTAATATTTCTG-3′) and 0363F1/R1 (5′-GGGGAAAACATATGAGGGAGATAACGCCGAGGAAG-3′/5′-AAAAGAATTCTCATTCGTCCTCCAGGCGTTCAAGG-3′). Using the NdeI–EcoRI restriction enzyme sites (indicated by underlines) incorporated during the PCR, the amplified fragments were inserted into the pET21a(+) expression vector (Novagen). After confirming the absence of unintended mutations, the plasmids were introduced into E. coli strain BL21-CodonPlus(DE3)-RIL. The transformants were cultivated at 37°C in LB medium with ampicillin until the optical density at 660 nm reached ∼ 0.5. Isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.1 mM to induce expression, and cells were cultivated for a further 4 h. Cells were harvested, resuspended in 50 mM Tris-HCl buffer (pH 8.0), and disrupted by sonication. After centrifugation (20 000 g, 20 min, 4°C), the soluble cell extracts were incubated at 80°C for 10 min. After removing thermolabile proteins deriving from the host by centrifugation (20 000 g, 20 min, 4°C), the supernatants were applied to anion-exchange chromatography (HiTrap Q HP, GE Healthcare, Little Chalfont, Buckinghamshire, UK), and proteins were eluted with a linear gradient of NaCl (0–1.0 M) in 50 mM Tris-HCl (pH 8.0) at a flow rate of 2.5 ml min−1. After concentrating the sample and exchanging the buffer to 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl using Amicon Ultra-4 10 K (Millipore, Billerica, MA), the samples were applied to a Superdex200 10/300 gel filtration column (GE Healthcare) with a mobile phase of 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl at a flow rate of 0.8 ml min−1. The same column and conditions were used to examine the molecular mass of the recombinant TK1968 protein, with the size markers aldolase (158 kDa), conalbumin (75 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa). Protein concentration was determined with the Protein Assay System (Bio-Rad, Hercules, CA) using bovine serum albumin as a standard.

Preparation of T. kodakarensis cell-free extracts

For preparation of cell-free extracts of the KUW1 and ΔTK1968 strains, cells were cultivated in ASW-YT-Pyr medium in the presence or absence of 1 mM CoA for 12 h at 85°C. After harvesting, cells were resuspended in 50 mM MES-NaOH (pH 6.4 at 70°C) and disrupted by sonication. After centrifugation (20 000 g, 20 min, 4°C), the supernatant was used to measure enzyme activity.

Examination of ketopantoate reductase activity of the TK1968 protein

KPR activity was measured by monitoring the decrease in absorption at 340 nm deriving from NAD(P)H using a spectrophotometer. Unless mentioned otherwise, the KPR reaction mixture contained 0.2 mM 2-oxopantoate, 0.2 mM NAD(P)H (Oriental Yeast, Tokyo, Japan), 1.1 μg ml−1 recombinant TK1968 protein and 50 mM MES-NaOH (pH 6.4 at 70°C). 2-Oxopantoate was prepared by hydrolysing 2-oxopantolactone (Sigma-Aldrich, St. Louis, MO) in 0.4 M NaOH for 1 h at 95°C. After pre-incubation of the reaction mixture without 2-oxopantoate for 2 min at the desired temperature, the reaction was initiated by the addition of 2-oxopantoate. The decrease in absorption at 340 nm was measured consecutively and rates were calculated. The rate of decrease in NAD(P)H in a reaction mixture without the TK1968 protein was subtracted from each assay result. When we performed a kinetic analysis for the forward reaction, the concentrations of the recombinant TK1968 protein were 0.13–1.1 μg ml−1. Kinetic parameters for 2-oxopantoate and NAD(P)H were determined using the standard method at 70°C with various concentrations of 2-oxopantoate (with 0.2 mM NADH) and NAD(P)H (with 0.2 mM 2-oxopantoate). For the reverse reaction, kinetic parameters for d-pantoate, l-pantoate and NAD+ were determined at 70°C with varying concentrations of d- or l-pantoate (with 1 mM NAD+) and NAD+ (with 1 mM d-pantoate). The reaction mixture contained d- or l-pantoate, NAD+ (Oriental Yeast), 0.13–1.1 μg ml−1 TK1968 protein and 50 mM MES-NaOH (pH 6.4 at 70°C). d-pantoate and l-pantoate were prepared by hydrolysing d-pantolactone and l-pantolactone (Sigma-Aldrich), respectively, in 0.4 M NaOH for 1 h at 95°C. After the reaction mixture was pre-incubated without pantoate for 2 min at 70°C, the reaction was initiated by the addition of d- or l-pantoate. The increase in absorption at 340 nm was measured consecutively. The rate of increase in NADH in a reaction mixture without TK1968 protein was subtracted from each assay result. When we examined the effects of CoA and acetyl-CoA, KPR activity was examined using the standard method at 70°C. When we examined the ketopantoate reductase activity in the cell-free extracts of the KUW1 and the ΔTK1968 strains, the reaction mixture contained 0.2 mM 2-oxopantoate, 0.2 mM NAD(P)H, 220∼240 μg ml−1 cell-free extracts and 50 mM MES-NaOH (pH 6.4 at 70°C) in the presence or absence of 1 mM CoA. The background rate NAD(P)H consumption in a reaction mixture without 2-oxopantoate was subtracted from each assay result. Kinetic parameters were calculated with IGOR Pro Ver. 5.03 (Wave-Metrics, Lake Oswego, OR).

Thermostability and effects of pH and temperature

For examining thermostability, the purified TK1968 protein (0.53 mg ml−1) in 50 mM Tris-HCl (pH 8.0) was incubated for various periods of time at 60°C, 70°C, 80°C or 90°C. After the protein solutions were cooled on ice for 30 min, KPR activity was measured with the standard method. In order to examine the effects of pH, the KPR reaction was performed at various pH values using the following buffers at 50 mM: MES (pH 5.2–7.1), piperazine-1,4-bis(2-ethanesulphonic acid) (PIPES) (pH 7.0–7.9), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (HEPES) (pH 7.2–8.2). The reactions were performed at 70°C, and the pH values of the buffers used represent those at 70°C. In order to examine the effects of temperature, the KPR reaction was carried out at various temperatures with the standard method. The data obtained were used to make an Arrhenius plot. The rate constant k (s−1) was calculated for each temperature, based on the equation V = k[ES] ≈ Vmax = k[E]0, assuming that the observed velocities were almost equal to Vmax, as substrate concentrations were well above their respective Km values. Here, V (μmol s−1), [ES] (μmol) and [E]0 (μmol) represent the observed initial velocity, the concentration of enzyme-substrate complexes and the initial concentration of enzyme respectively.

Substrate specificity of KPR for 2-oxo acids

In order to examine the substrate specificity of the TK1968 protein for 2-oxo acids, 1 or 10 mM of 2-oxopantoate, pyruvate, oxaloacetate or 2-oxoglutarate was added to the reaction mixture at 70°C.

Examination of ketopantoate hydroxymethyltransferase activity of TK0363

KPHMT activity was examined by measuring the rate of 2-oxopantoate formation by HPLC. The standard KPHMT reaction mixture contained 50 mM 2-oxoisovalerate (Sigma-Aldrich), 2 mM formaldehyde or N5, N10-methylenetetrahydrofolate, 3.4 μg ml−1 recombinant TK0363 protein, 10 mM MgCl2 and 50 mM MES-NaOH (pH 6.5 at 85°C). Kinetic parameters for 2-oxoisovalerate and formaldehyde were determined using the standard method at 85°C with various concentrations of 2-oxoisovalerate (with 2 mM N5, N10-methylenetetrahydrofolate) and formaldehyde (with 50 mM 2-oxoisovalerate). When the effect of CoA was examined, the reaction mixture contained 5 mM 2-oxoisovalerate, 2 mM N5, N10-methylenetetrahydrofolate, 3.4 μg ml−1 recombinant TK0363 protein, 10 mM MgCl2 and 50 mM MES-NaOH (pH 6.5 at 85°C). N5, N10-Methylenetetrahydrofolate was prepared by adding equimolar formaldehyde to tetrahydrofolate (Sigma-Aldrich) solution at pH 9.5, in which N5, N10-methylenetetrahydrofolate is reported to be stable (Osborn et al., 1960; Sobti et al., 2000). The reaction was initiated by the addition of formaldehyde or N5, N10-methylenetetrahydrofolate, and incubated at 85°C for 10, 20 and 30 min. The reaction was stopped by cooling the mixture on ice. The recombinant TK0363 proteins were removed by ultrafiltration with Amicon Ultra-0.5 10K (Millipore), and 10 μl aliquots were applied to a COSMOSIL 5C18-PAQ column. Compounds were separated with 50 mM NaH2PO4 in water at a flow rate of 1.0 ml min−1 and detected by measuring absorbance at 210 nm. The amount of 2-oxopantoate generated in the reactions after 10, 20 and 30 min was measured and used to calculate the reaction rate. The relationship between the amount of 2-oxopantoate and the peak area was determined beforehand by examining the peak areas of various amounts of 2-oxopantoate. A linear increase in 2-oxopantoate was observed under these conditions, allowing us to calculate the initial velocity of the KPHMT reaction. When we examined the effect of CoA, KPHMT activity was examined using the standard method at 85°C.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was partially supported by Grant-in-Aid for the Japan Society for the Promotion of Science (JSPS) fellows (Grant No. 24.2716) to H.T.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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