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Keywords:

  • C1-metabolism;
  • cyclohydrolase;
  • methanogenic archaea;
  • methylotrophic bacteria;
  • tetrahydrofolate;
  • tetrahydromethanopterin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units. In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U·mg−1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U·mg−1). Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence. The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U·mg−1 (Vmax = 1500 U·mg−1; Km = 30 µm) was found to be composed of two identical subunits of molecular mass 33 kDa. Its sequence was ≈ 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea. The methenyl H4F cyclohydrolase with a specific activity of 100 U·mg−1 (Vmax = 330 U·mg−1; Km = 80 µm) was found to be composed of two identical subunits of molecular mass 22 kDa. Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases. Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M. extorquens AM1.

Abbreviations
Fch

methenyl H4F cyclohydrolase

H4F

tetrahydrofolate

H4MPT

tetrahydromethanopterin

Mch

methenyl H4MPT cyclohydrolase

MFR

methanofuran.

Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate (H4F) analog (for structures see Fig. 1) previously thought to occur only in the anaerobic methanogenic and sulfate-reducing archaea [1,2] but recently also found, in a dephospho form, in the aerobic methylotrophic proteobacterium Methylobacterium extorquens AM1 [3]. The latter organism was shown to contain both dephospho H4MPT and H4F at comparable concentrations [4].

image

Figure 1. Structures of tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F). The H4MPT derivative found in M. extorquens AM1 lacks the α-hydroxyglutaryl phosphate unit [3]. The dephospho H4MPT is also found in methanogenic archaea [25].

H4MPT and H4F both carry one-carbon units at the oxidation levels of formate, formaldehyde and methanol. These one-carbon units are bound to N5 (N5-formyl, N5-methyl), N10 (N10-formyl) or both N5 and N10 (N5,N10-methenyl, N 5,N10-methylene) of the two reduced pterins. Functionally, the most important structural difference between H4MPT and H4F is that H4MPT has an electron-donating methylene group in conjugation with N10 via the aromatic ring, whereas H4F has an electron-withdrawing group in this position. The pKa of the para amino group has been estimated to be +2.4 in H4MPT and −1.2 in H4F [5]. As a result the redox potential Eo′ = –320 mV of the methylene H4MPT/methyl H4MPT couple is 120 mV more negative than the Eo′ = –200 mV of the methylene H4F/methyl H4F couple, and Eo′ = – 390 mV of the methenyl H4MPT/methylene H4MPT couple is 90 mV more negative than the Eo′ = –300 mV of the methenyl H4F/methylene H4F couple [5]. These differences may help explain why some organisms use H4MPT, some H4F and some both H4MPT and H4F in their C1 metabolism. Thus from the redox potentials and Eo′ = –320 mV of the NAD(P)/NAD(P)H couple it can be predicted that the oxidation of C1 units with NAD(P) is thermodynamically favored if the C1 unit is bound to H4MPT and the reduction of C1 units with NAD(P)H is favored if the C1 unit is bound to H4F. It has therefore recently been proposed that in M. extorquens AM1 H4MPT is involved in the oxidation of methanol to CO2, whereas H4F could be involved in the reduction of CO2 to the oxidation level of formaldehyde [4].

M. extorquens AM1 has been shown to contain two methylene tetrahydropterin dehydrogenases, one of which is NADP dependent and the other NAD dependent. The NADP-dependent dehydrogenase was purified and characterized. The purified enzyme catalyzed the NADP-dependent dehydrogenation of methylene H4MPT and also that of methylene H4F. With methylene H4F as substrate, however, the specific activity and catalytic efficiency were ≈ 20 fold lower than with methylene H4MPT. It was identified as the mtdA gene product by its N-terminal amino acid sequence [4]. The NAD-dependent methylene tetrahydropterin dehydrogenase has not yet been purified. The activity can be separated from the NADP-dependent activity by ammonium sulfate precipitation [4]. There is genetic evidence that the NAD-dependent enzyme is encoded by the orfX gene [3].

In this paper we report that M. extorquens AM1 also contains two methenyl tetrahydropterin cyclohydrolases catalyzing reactions 1 and 2, respectively.

  • Methenyl H4MPT++H2O  ⇌ N5-formyl H4MPT+H+(1)
  • image
  • Methenyl H4F++H2O  ⇌ N10-formyl H4F+H+(2)
  • image

The two enzymes were purified and characterized.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Coenzymes

Methenyl H4MPT was purified from Methanobacterium thermoautotrophicum strain Marburg (DSM 2133) [8]. Dephospho H4MPT was isolated as described previously [4]. Methenyl H4F was generated from methylene H4F by dehydrogenation via NADP-dependent methylene H4MPT dehydrogenase from M. extorquens AM1 and purified by HPLC [4]. Methylene H4F was generated from H4F (Sigma) and formaldehyde by spontaneous reaction.

Growth of bacteria

M. extorquens AM1 was grown on methanol (100 mm) at 30 °C in minimal medium as described previously [9]. The cultures were harvested in the late-exponential phase at a cell concentration of 3 g (wet mass)·L−1. Cells were pelleted by centrifugation at 5000 g and stored at −20 °C.

Preparation of cell extracts

Frozen cells (10 g) were suspended in 16 mL of anoxic 50 mm Mops/KOH pH 7.0 containing 2 mm dithiothreitol and 2 mg of DNase I, and passed three times through a French pressure cell at 1.2 × 108 Pa under anaerobic conditions. The soluble fraction of the cell extract (containing 95% of the cyclohydrolase activities) was obtained by ultracentrifugation at 150 000 g for 1 h. The protein concentration was determined with the Bradford assay method [10] using the Biorad reagent with BSA as standard.

Determination of enzyme activities

The assays were performed routinely at 30 °C in 1-mL cuvettes (d = 1 cm) in a total volume of 0.7 mL. Methenyl H4MPT cyclohydrolase activity was followed photometrically by measuring the decrease in absorbance at 335 nm (ε = 21.6 mm−1·cm−1) [1]. The standard assay contained 50 mm tricine/KOH pH 8.0, 0.6 m NaCl and 25 µm methenyl H4MPT. The reaction was started by the addition of protein. Methenyl H4F cyclohydrolase activity was determined by measuring the decrease in absorbance at 356 nm (ε = 24.9 mm−1·cm−1). The standard assay contained 0.5 m triethanolamine/HCl pH 7.5 and 25 µm methenyl H4F. The reaction was started by the addition of protein. The rate of spontaneous hydrolysis of methenyl H4F was considered. One unit of enzyme activity is defined as the hydrolysis of 1 µmol methenyl H4MPT/methenyl H4F. Phosphoglycerate kinase [11] and formimino H4F cyclodeaminase [12] were assayed as described.

Purification of methenyl H4MPT cyclohydrolase

All purification steps were performed under anaerobic conditions. The soluble fraction of the cell extract (20 mL) was loaded on DEAE–Sephacel (Sigma; 26/10) equilibrated with 50 mm Mops/KOH pH 7.0 containing 2 mm dithiothreitol (buffer A). Protein was eluted with NaCl gradients in buffer A, 50 mL 0 m NaCl, 5 mL 0–0.16 m NaCl, 40 mL 0.16 m NaCl, 90 mL 0.2 m NaCl, 160 mL 0.2–0.5 m NaCl, 5 mL 0.5–1 m NaCl, 50 mL 1–2 m NaCl and 30 mL 2 m NaCl. Methenyl H4MPT cyclohydrolase was eluted with 1 m NaCl (60 mL). The most active fractions (48 mL) were applied to Q Sepharose (High Performance 16/10, Pharmacia Biotech) equilibrated with buffer A. Protein was eluted with a NaCl step gradient, 20 mL 0.14 m NaCl, 230 mL 0.14–0.6 m NaCl and 20 mL 0.6–2 m NaCl. The enzyme activity was recovered at 0.4 m NaCl (12 mL). Protein was subjected to chromatography on Uno Q (Uno Q-6, Biorad) equilibrated with buffer A and eluted using NaCl gradients, 40 mL 0.1–0.5 m NaCl, 2 mL 0.5–1 m NaCl and 5 mL 1–2 m NaCl. Methenyl H4MPT cyclohydrolase was eluted with 0.3 m NaCl (1.5 mL).

Purification of methenyl H4F cyclohydrolase

All purification steps were performed under anaerobic conditions. The soluble fraction of the cell extract (20 mL) was loaded onto DEAE–Sephacel (Sigma; 26/10) and eluted as described above. Methenyl H4F cyclohydrolase was eluted with 0.2 m NaCl (54 mL) in 50 mm Mops/KOH pH 7.0 containing 2 mm dithiothreitol (buffer A). The most active fractions (60 mL) were applied to Q Sepharose (High Performance 16/10, Pharmacia Biotech) equilibrated with buffer A. Protein was eluted using a linear increasing NaCl gradient in buffer A from 0 to 0.6 m NaCl in 250 mL. Methenyl H4F cyclohydrolase was eluted with 0.22 m NaCl (15 mL) and was subjected to chromatography on HA-Ultrogel (Biorad; 16/10) equilibrated with 10 mm potassium phosphate pH 7.0. Protein was eluted using a linear increasing NaCl gradient in buffer A from 10 to 500 mm potassium phosphate in 250 mL. Methenyl H4F cyclohydrolase was eluted with 90 mm potassium phosphate (250 mL). Protein was loaded onto Mono Q (Mono Q HR 10/10, Pharmacia Biotech) equilibrated with buffer A and eluted using a linear increasing NaCl gradient in buffer A from 0 to 2 m NaCl in 160 mL. Methenyl H4F cyclohydrolase eluted with 0.2 m NaCl (6 mL). Protein was further chromatographied on Uno Q (Uno Q-6, Biorad) equilibrated with buffer A using a linear increasing NaCl gradient from 0 to 2 m NaCl in 45 mL. Methenyl H4F cyclohydrolase was eluted with 0.2 m NaCl (0.5 mL).

Determination of the N-terminal amino acid sequences

Purified enzymes were electrophoresed in the presence of SDS and electroblotted onto a poly(vinyl trifluoride) membrane (Applied Biosystems). Sequence determination was performed on a 477 protein/peptide sequencer from Applied Biosystems by Dr D. Linder, Giessen.

Construction of insertion mutants in fchA

Insertion mutations in the two sites (BspMI and AccI) of fchA (orf4) were constructed previously. The corresponding donor strains were used to generate new mutants as described previously [13].

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell extracts of methanol-grown M. extorquens AM1 catalyzed the hydrolysis of methenyl H4MPT at a specific activity of 0.9 U·mg−1 protein and the hydrolysis of methenyl H4F at a specific activity of 0.23 U·mg−1 protein. The two activities, which were associated with the soluble cell fraction, could be separated by chromatography on DEAE–Sephacel (Fig. 2).

image

Figure 2. Separation of the methenyl H4MPT cyclohydrolase activity (Mch) from the methenyl H4F cyclohydrolase activity (Fch) in cell extracts ofM. extorquensAM1 by chromatography on DEAE–Sephacel. Cell extract (20 mL) of M. extorquens AM1 was loaded onto a DEAE–Sephacel column (2.6 cm × 12 cm) and eluted using a stepwise increasing sodium chloride gradient (···). Eluting protein was measured at 280 nm (—). The enzyme activity profile of Mch (•–•) and Fch (▪–▪) is shown.

Methenyl H4MPT cyclohydrolase

Purification of the enzyme was achieved under anaerobic conditions as described in Table 1. Purification was 700-fold with a 16% yield. If during the purification anaerobic conditions were not employed, the activity yield was much lower.

Table 1. Purification of methenyl H4MPT cyclohydrolase fromM. extorquensAM1 grown on methanol.
Purification stepsProtein (mg)Activity (U)Specific activity (U·mg−1)Purification (-fold)Yield (%)
  1. The enzyme purification was performed under anaerobic conditions. Enzyme activities were determined at 30 °C under standard assay conditions.

Cell extract4654000.91100
DEAE–Sephacel312628.51066
Q-Sepharose1.319215517248
Uno Q0.16363070016

SDS/PAGE revealed the presence of only one polypeptide of apparent molecular mass 33 kDa (Fig. 3A). The native enzyme migrated in polyacrylamide gradient gels with an apparent molecular mass of 67 kDa indicating that the methenyl H4MPT cyclohydrolase is a homodimer (Fig. 3B). The ultraviolet/visible spectrum of the purified enzyme was very similar to that of albumin, indicating the absence of a chromophoric prosthetic group.

image

Figure 3. Electrophoretic properties of purified methenyl H4MPT cyclohydrolase from M. extorquens AM1 under denaturating and nondenaturating conditions. (A) SDS/PAGE. Protein was separated on a 16% polyacrylamide gel and subsequently stained with Coomassie brilliant blue. Lanes 1 and 4, low molecular mass standards (Pharmacia Biotech); lane 2 and lane 3, 6 µg and 3 µg of purified Mch, respectively. (B) Native polyacrylamide gradient gel electrophoresis. Protein was separated in a polyacrylamide gel (4–15%) (Biorad) and subsequently stained with Coomassie brilliant blue. Lane 1, molecular mass standard; lane 2, 3 µg of purified Mch.

The N-terminal amino acid sequence was determined to be MSSNTSAPSLNALAG. This matches exactly that predicted for the orfZ gene product [3]. Its sequence is ≈ 40% identical and its molecular mass is in agreement with that of methenyl H4MPT cyclohydrolases from methanogenic archaea [14]. M. extorquens AM1 with an insertion mutation (single crossover) in orfZ was shown to lack the ability to grow on methanol [3].

The purified cyclohydrolase catalyzed the hydrolysis of methenyl H4MPT to N5-formyl H4MPT rather than to N 10-formyl H4MPT as evidenced by the ultraviolet/visible spectrum of the product (Fig. 4). Methenyl H4F was not hydrolyzed. The enzyme did not catalyze the dehydrogenation of methylene H4MPT with either NAD(P) or coenzyme F420 as electron acceptor. Like the cyclohydrolase from methanogenic archaea [14,15], the methenyl H4MPT cyclohydrolase from M. extorquens AM1 is therefore specific for H4MPT, catalyzes the formation of the N5-derivative and is a monofunctional enzyme.

image

Figure 4. Ultraviolet/visible spectra of the product formed (A) by enzyme catalyzed hydrolysis of methenyl H4MPT at pH 8.0 and (B) by hydrolysis of methenyl H4MPT at pH 13. Spectrum A is characteristic for N5-formyl H4MPT and spectrum B for N10-formyl H4MPT [21]. The reactions were performed under standard assay conditions in a volume of 50 µL. The reaction was started by the addition of 60 ng purified Mch or 2 µL 2 m NaOH. The absorption spectra were recorded every 5 s.

The cyclohydrolase was routinely assayed at 30 °C in 50 mm tricine/KOH pH 8.0 containing 25 µm methenyl H4MPT and 0.6 m NaCl. Under these assay conditions the purified enzyme exhibited a specific activity of 630 U·mg−1 protein. When tested with dephospho methenyl H4MPT (25 µm) isolated from M. extorquens AM1 the specific activity was 1470 U·mg−1 protein. The Km values for methenyl H4MPT and for dephospho methenyl H4MPT were found to be identical at 30 µm. With dephospho methenyl H4MPT as substrate the Vmax value (3500 U·mg−1) was, however, 2.3 times higher than with methenyl H4MPT (1500 U·mg−1).

The pH optimum for the catalysis of methenyl H4MPT hydrolysis was found to be near 8.5 and the temperature optimum near 40 °C.

For optimal activity the enzyme required the presence of salt, which is why the standard assay contained 0.6 m NaCl. When the salt was omitted the specific activity was only 15% of that in the presence of salt. Salt also increased the heat stability of the enzyme. In the absence of salt the enzyme lost 50% of its activity within 10 min when incubated at 60 °C in 50 mm tricine/KOH pH 8.5. In the presence of salt (1 m K2HPO4) no activity was lost within 25 min

Methenyl H4F cyclohydrolase

The enzyme was purified 450-fold in a 10% yield (Table 2). Five chromatographic steps were required to separate the cyclohydrolase from phosphoglycerate kinase, which copurified with the cyclohydrolase in most of the steps. The two activities could, however, be separated by chromatography on Uno Q, but even in this step separation was not complete. The final preparation obtained showed 104 U·mg−1 methenyl H4F cyclohydrolase activity and 1.8 U·mg−1 phosphoglycerate kinase activity. The specific activity of phosphoglycerate kinase in the cell extract was 0.02 U·mg−1.

Table 2. Purification of methenyl H4F cyclohydrolase fromM. extorquensAM1 grown on methanol.
Purification stepsProtein (mg)Activity (U)Specific activity (U·mg−1)Purification (-fold)Yield (%)
  1. The enzyme purification was performed under anaerobic conditions. Enzyme activities were determined at 30 °C under standard assay conditions.

Cell extract5171190.231100
DEAE–Sephacel102590.582.550
Q-Sepharose12262.29.722
HA-Ultrogel1.1518166815
Mono Q0.459.320.5897.8
Uno Q0.111210445010

SDS/PAGE revealed the presence in the preparation of two polypeptides, a major one with an apparent molecular mass of 22 kDa and a minor one with an apparent molecular mass of 43 kDa (Fig. 5A). The 43 kDa polypeptide was identified via its N-terminal amino acid sequence (GDFRTLDDAGPLQGXRVLLR) to be phosphoglycerate kinase (60% sequence identity to the enzyme from Zymomonas mobilis; [16]). The native enzyme migrated in polyacrylamide gradient gels with an apparent molecular mass of 45 kDa indicating that the methenyl H4F cyclohydrolase has a homodimeric structure (Fig. 5B). The ultraviolet/visible spectrum of the purified enzyme showed no signs for the presence of a chromophoric prosthetic group.

image

Figure 5. Electrophoretic properties of purified methenyl H4F cyclohydrolase fromM. extorquensAM1 under denaturating and non denaturating conditions. (A) SDS/PAGE. Protein was separated on a 16% polyacrylamide gel and subsequently stained with Coomassie brilliant blue. Lane 1, low molecular mass standards (Pharmacia Biotech); lane 2, 5 µg of purified Fch. (B) Native polyacrylamide gradient gel electrophoresis. Protein was separated in a polyacrylamide gel (4–15%) (Biorad) and subsequently stained with Coomassie brilliant blue. Lane 1, molecular mass standard; lane 2, 5 µg of purified Fch.

The N-terminal amino acid sequence of the 22 kDa polypeptide was determined to be MAGNETIETFLDGLASSAPTP. It matched exactly that predicted for the orf4 gene product [13]. We now redesignate the gene as fchA (for folate cyclohydrolase). The sequence of FchA is not similar to that of methenyl H4MPT cyclohydrolase or to that of other methenyl H4F cyclohydrolases. There is a sequence similarity (34% identity) to porcine formimino H4F cyclodeaminase which catalyzes the formation of methenyl H4F and NH3 from N5-formimino H4F [12]. However, formimino H4F cyclodeaminase activity could not be detected in Fch preparations.

Mutants in orf4 were obtained previously with low frequency and found to be double crossovers negative for growth on C1 and C2 compounds [13]. However, it was not possible to complement them with the corresponding DNA fragments. In this study we therefore selected new mutants in fchA (orf4), again with very low frequency, using both donors constructed in the previous study. All of the mutants obtained were the result of single crossover events, and these could still grow on methanol (data not shown). It was not possible to isolate double crossover (null) mutants on succinate, which might indicate that fchA has an essential function in the central metabolism of M. extorquens AM1. DNA–DNA (Southern) hybridization and PCR analysis confirmed that these mutants carried a copy of intact fchA and a copy of fchA interrupted with the kanamycin resistance gene (data not shown).

The purified cyclohydrolase catalyzed the hydrolysis of methenyl H4F to N10-formyl H4F rather than to N5-formyl H4F as indicated by the ultraviolet/visible spectrum of the product (Fig. 6). Methenyl H4MPT was not a substrate. With respect to the formation of the N10-formyl derivative and to the specificity, the enzyme is thus like all other known methenyl H4F cyclohydrolases. The cyclohydrolase did not exhibit methylene H4F dehydrogenase activity and is therefore probably a monofunctional enzyme. In this respect the enzyme differs from most characterized methenyl H4F cyclohydrolases, which are generally multifunctional enzymes [17].

image

Figure 6. Ultraviolet/visible spectrum of the product formed by the enzyme catalyzed hydrolysis of methenyl H4F at pH 6. The spectrum is characteristic for N10-formyl H4F rather than for N5-formyl H4F [26]. The reaction was performed under standard assay conditions in 120 mm potassium phosphate pH 6 in a volume of 50 µL. The reaction was started by the addition of 20 ng purified Fch. The absorption spectra were recorded every 5 s.

The methenyl H4F cyclohydrolase activity was routinely assayed at 30 °C in 50 mm triethanolamine/HCl pH 7.5 containing 25 µm methenyl H4F. Under these assay conditions the purified enzyme exhibited a specific activity of 104 U·mg−1. The Km for methenyl H4F was determined to be 80 µm and Vmax to be 330 U·mg−1. The methenyl H4F activity was not stimulated by salts.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The results presented here indicate that cells of M. extorquens AM1 contain a methenyl H4MPT cyclohydrolase and a methenyl H4F cyclohydrolase. The specific activity of the H4MPT specific enzyme was four times higher in the cell extract than that of the H4F specific enzyme. When tested with dephospho H4MPT, which is the tetrahydromethanopterin derivative present in M. extorquens AM1, the specific activity of the methenyl H4MPT cyclohydrolase was 10 times as high as that of the methenyl H4F cyclohydrolase. This might suggest a catabolic function for the H4MPT-specific cyclohydrolase and an anabolic function for the H4F specific enzyme as recently proposed by Vorholt et al. [4]. In addition it is possible that both pathways could function in formaldehyde detoxification.

The methenyl H4MPT cyclohydrolase was found to catalyze the formation of N5-formyl H4MPT as product and the methenyl H4F cyclohydrolase to catalyze the formation of N10-formyl H4F. The two products differ considerably in their hydrolysis potential. Whereas the hydrolysis of N10-formyl H4F is associated with a free energy change ΔGo′ of −23.4 kJ·mol−1[18] that of N5-formyl H4MPT, and probably also of N5-formyl H4F, is only exergonic by −8 kJ·mol−1[estimated from ΔGo′ = –4 kJ·mol−1 for the formyltransfer from formyl methanfuran (MFR) to H4MPT, reaction 3, ΔGo′ = +4 kJ·mol−1 for CO2 reduction with H2 to formate and ΔGo′ = +16 kJ·mol−1 for CO2 reduction with H2 to formyl MFR]. The difference explains why in general in formyltransferase reactions only the N5-formyl derivatives are formed and why the formation of N10-formyl H4F requires ATP.

Cell extracts of M. extorquens AM1 were found to contain formyl MFR : H4MPT formyltransferase activity [3] and formyl H4F synthetase activity [19,20] catalyzing reactions 3 and 4, respectively. MFR is a furfurylamine derivative previously found only in methanogenic and sulfate-reducing archaea [1]; formyl MFR is the N-formyl furfurylamine derivative.

  • Formyl MFR+H4MPT ⇌ N5-formyl H4MPT+MFR(3)
  • image
  • Formate+ATP+H4F ⇌ N10-formyl H4F+ADP+Pi(4)
  • image

The free energy changes associated with these two reactions indicate that the formation of the formyl derivatives of H4MPT and H4F are thermodynamically favored, and indeed this direction is generally also the physiological one. There is, however, evidence that under some conditions the function of the two enzymes can also be to catalyze the reverse reactions. For formyl MFR : H4MPT formyltransferase this is the case in Methanosarcina species growing on methanol, methylamines or methylthiols [6] and for formyl H4F synthetase this is the case in some clostridia growing on purines [18].

To understand the function of the two cyclohydrolases in M. extorquens AM1 it is important to know that this organism also contains an NAD-dependent formate dehydrogenase [22] (catalyzing reaction 5), that there is genetic evidence for the presence of a formyl MFR dehydrogenase [3] (catalyzing reaction 6), and that these two enzymes can principally also function in both directions.

  • Formate +NAD ⇌ CO2+ NADH(5)
  • image
  • Formyl MFR ⇌ CO2+MFR+2[H](6)
  • image

Purification and characterization of the enzymes catalyzing reactions 3–6 and identification of their encoding genes will be the next steps in the elucidation of the different roles that H4MPT and H4F may play in the C1-unit metabolism of M. extorquens AM1. Also the presence of MFR or a MFR analog in M. extorquens AM1 remains to be demonstrated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by the Max-Planck-Gesellschaft, the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie (R.K.T.) and by an NIH grant (M.E.L.; GM36296).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
Footnotes
  1. Enzymes: NAD-dependent formate dehydrogenase (EC1.2.1.2); formylmethanofuran dehydrogenase (EC1.2.99.5); NAD(P)-dependent methylene tetrahydromethanopterin dehydrogenase (EC1.5.1.-); NADP-dependent methylene tetrahydrofolate dehydrogenase (EC1.5.1.5); NAD-dependent methylene tetrahydrofolate dehydrogenase (EC1.5.1.15); formylmethanofuran:tetrahydromethanopterin formyltransferase (EC2.3.1.101); methenyl tetrahydrofolate cyclohydrolase (EC3.5.4.9); methenyl tetrahydromethanopterin cyclohydrolase (EC3.5.4.27); formimino tetrahydrofolate cyclodeaminase (EC4.3.1.4); formyl tetrahydrofolate synthetase (EC6.3.4.3).