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Previous Article | Next Article 
Journal of Bacteriology, December 2000, p. 6645-6650, Vol. 182, No. 23
Max Planck Institute for Terrestrial
Microbiology, 35043 Marburg, Germany,1 and
Department of Microbiology2 and
Department of Chemical Engineering,3
University of Washington, Seattle, Washington 98195
Received 22 June 2000/Accepted 21 September 2000
Formaldehyde is toxic for all organisms from bacteria to humans due
to its reactivity with biological macromolecules. Organisms that grow
aerobically on single-carbon compounds such as methanol and methane
face a special challenge in this regard because formaldehyde is a
central metabolic intermediate during methylotrophic growth. In the
Formaldehyde is a highly reactive
chemical that exerts a toxic effect on organisms through its
nonspecific reactivity with proteins and nucleic acids (9,
11). All organisms produce low concentrations of formaldehyde as
a result of demethylation reactions (5, 13). Methylotrophic
bacteria represent an extreme case of formaldehyde handling, as aerobic
growth on single-carbon (C1) substrates such as methanol or
methane involves formaldehyde as a central metabolic intermediate
(22). A typical methylotrophic bacterium with a doubling
time of 2 h generates and consumes formaldehyde at a specific rate
of 0.5 mmol min The catabolism of C1 units is best studied in
Methylobacterium extorquens AM1. The
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Novel Formaldehyde-Activating Enzyme in
Methylobacterium extorquens AM1 Required for Growth on
Methanol
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-proteobacterium Methylobacterium extorquens AM1, we
found a previously unknown enzyme that efficiently catalyzes the
removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a
reaction which also proceeds spontaneously, but at a lower rate than
that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme
(Fae) was purified from M. extorquens AM1 and found to be
one of the major proteins in the cytoplasm. The encoding gene is
located within a cluster of genes for enzymes involved in the further
oxidation of methylene tetrahydromethanopterin to CO2. Mutants of M. extorquens AM1 defective in Fae were able to
grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this
enzyme are predicted to be encoded by uncharacterized genes from
archaea, indicating that this type of enzyme occurs outside the
methylotrophic bacteria.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 g1 (dry weight),
corresponding to 0.1 mmol per min and per ml of cytoplasmic volume
(1). Thus, if the consumption of formaldehyde were
inhibited, the cytoplasmic formaldehyde concentration would increase to
about 100 mM within less than 1 min. Therefore, the metabolism of
aerobic methylotrophic bacteria must be constructed and regulated such
that the rate of formaldehyde utilization does not become limiting; a
limitation would rapidly become detrimental.
-proteobacterium
M. extorquens AM1 is a facultative methylotroph that can
grow aerobically on methanol and methylamine but also on some
multicarbon substrates such as succinate. A scheme of its metabolism
during growth on methanol is shown in Fig.
1. Methanol is oxidized to formaldehyde
in the periplasm in a reaction catalyzed by a pyrroloquinoline
quinone-dependent methanol dehydrogenase. The formaldehyde then crosses
the cytoplasmic membrane, and in the cytoplasm it then reacts with the
C1 carrier molecules tetrahydrofolate (H4F) and
tetrahydromethanopterin (H4MPT) (7).
H4MPT is an H4F analogue in methanogenic and
sulfate-reducing archaea and was also recently found in most
methylotrophic bacteria including methanotrophic bacteria
(34). Based on the specificity and activity of the
respective C1 unit-converting enzymes, we have proposed
that, in M. extorquens AM1, the H4MPT-dependent pathway is the primary formaldehyde oxidation pathway and the H4F-dependent pathway is mainly involved in formaldehyde
assimilation (33) which further proceeds by the serine cycle
(22) (Fig. 1). A central reaction in the metabolism of
methanol in M. extorquens AM1 is the condensation of
formaldehyde with H4MPT and H4F, resulting in
the formation of the
N5,N10-methylene
derivatives:
These reactions are known to occur spontaneously (8, 16, 20,
25). However, the question arises whether the spontaneous rates
are sufficiently high to accommodate the rate of formaldehyde production, as has been discussed before with respect to methylene H4F formation by Attwood and Quayle (1). Studies
of nonmethylotrophic organisms suggested that the condensation of
formaldehyde and H4F is nonenzymatic (15-17).
We have now reinvestigated this problem with a methylotrophic
bacterium, which, as pointed out above, must maintain a high rate of
formaldehyde consumption inside the cell. We did not find a
formaldehyde-H4F condensing activity. However, we
discovered and purified an enzyme that catalyzes the condensation of
formaldehyde and H4MPT to methylene H4MPT and have shown that it is required for growth of M. extorquens
AM1 on methanol and involved in formaldehyde detoxification.
View larger version (21K):
[in a new window]
FIG. 1.
C1 metabolism of M. extorquens
AM1. Formaldehyde is produced in the periplasm of the cell from
methanol by a pyrroloquinoline quinone-dependent methanol dehydrogenase
and crosses the membrane. Cytoplasmic formaldehyde reacts with either
H4F or H4MPT. The condensation of formaldehyde
and H4MPT is catalyzed by Fae (this study). The
C1 unit is utilized for further oxidation to
CO2 or incorporated via the serine cycle. a1,
NADP+-dependent methylene H4F dehydrogenase
MtdA (33); a2, methenyl H4F
cyclohydrolase FchA (27); a3, formyl
H4F synthase; a4, formate dehydrogenase;
b1, NAD(P)+-methylene H4MPT
dehydrogenases MtdA and MtdB (12, 33); b2,
methenyl H4MPT cyclohydrolase Mch (27);
b3, formyl methanofuran:H4MPT
formyltransferase; b4, formyl methanofuran dehydrogenase.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Coenzymes. H4MPT was purified from Methanobacterium thermoautotrophicum Marburg (DSM 2133) (3). H4F was purchased from Sigma. Anoxic stock solutions of H4MPT and H4F were prepared in 50 mM Tricine-KOH (pH 7.0).
Growth of organisms.
M. extorquens AM1 was grown on
methanol (100 mM) or succinate (20 mM) at 30°C in minimal medium as
described previously (10). The cultures were harvested in
the late exponential phase at a cell concentration of 3 g of wet
mass/liter. Cells were pelleted by centrifugation at 5,000 × g and stored at 
20°C. M. thermoautotrophicum Marburg was grown at 65°C on H2-CO2 (80/20)
in a minimal medium (30). Methanosarcina barkeri
Fusaro (DSM 804) was grown at 37°C on methanol (18). Cells
of the methanogenic archaea were harvested anaerobically in the late
exponential phase.
Preparation of cell extracts. Cells were disrupted in a French press, and cell extracts were prepared as described before (33). Protein concentration was determined by the Bradford assay (2) by using the Bio-Rad reagent with bovine serum albumin as the standard.
Determination of the activity of Fae.
Formaldehyde-activating enzyme (Fae) activity was routinely assayed at
30°C and pH 8.0. The standard assay mixture contained 50 mM
Tricine-KOH, 30 mM MgCl2, 50 µM H4MPT
(isolated from M. thermoautotrophicum) or 50 µM
H4F, enzyme, and 1.6 mM formaldehyde. The reaction was
started with formaldehyde. The formation of methylene H4MPT
was monitored by measuring the increase in absorbance at 250 nm using a
250 of 8.5 mM1 cm1, and
the formation of methylene H4F was monitored by measuring the increase in absorbance at 295 nm using a 295 of
3.0 mM1 cm1. A unit of activity was defined
as the formation of 1 µmol of methylene H4MPT from
H4MPT and formaldehyde per min minus the spontaneous
reaction rate without enzyme added.
Purification of Fae from M. extorquens AM1. Cell extracts of M. extorquens AM1 were ultracentrifuged (150,000 × g for 1 h), and the soluble fraction was loaded onto DEAE-Sephacel (26/10; Sigma) columns equilibrated with 50 mM MOPS (morpholine propanesulfonic acid)-KOH, pH 7.0 (buffer A). Protein was eluted with NaCl in buffer A-80 ml of 0 M NaCl-120 ml of 0.1 M NaCl-200 ml of 0.1 to 0.4 M NaCl-100 ml of 0.5 M NaCl. Fae was eluted with about 0.3 M NaCl and applied to a Q-Sepharose (High Performance 16/10; Amersham Pharmacia Biotech) column equilibrated with buffer A. Protein was eluted with a linearly increasing gradient of 0 to 0.3 M NaCl within 450 ml. Fae eluted at 0.13 M NaCl and was subjected to chromatography on hydroxylapatite (16/10; Bio-Rad) equilibrated with 10 mM potassium phosphate, pH 7.0. Protein was eluted with a step gradient of 50, 100, 150, 175, and 200 mM potassium phosphate (25 ml each step). Fae was eluted at 175 mM potassium phosphate. The purification was performed under anoxic conditions. Although the enzyme from M. extorquens AM1 was found to be oxygen tolerant, the anoxic preparation turned out to be more suitable and to yield purer enzyme preparations.
Construction and analysis of a Fae mutant strain. A 2.0-kb chromosomal region of M. extorquens AM1 containing fae (accession no. AF032114) (7) was amplified using the primers CM-faef, 5'-GTCCCAAATCGATGACGAAG-3', and CM-faer, 5'-GGTTCACGCGATGTCTCAC-3'. The resulting PCR product was cloned into the pCR2.1 TOPO TA cloning vector (Invitrogen) to yield pCM112. The 2.1-kb BamHI-SphI region of pCM112 was subcloned into pUC19 (36), generating pCM113. The kanamycin resistance gene from pUC4K (32) was then inserted between the two HincII sites found in fae, in the same direction as fae is transcribed, as the direction is known, to create pCM114. The 3.1-kb BamHI-SphI fragment of pCM114 containing the fae::kan allele was then excised, blunted with T4 DNA polymerase, and inserted into the SmaI site of the suicide vector pAYC61 (6) to yield pCM115. Conjugation into M. extorquens AM1 and selection of exconjugates were performed as described previously (6). PCR analysis and antibiotic resistance phenotype confirmed that strain CM115.1 contains a single, interrupted copy of fae generated by allelic exchange. Growth of CM115.1 (fae::kan) on various media was compared to that of a kanamycin-resistant wild-type strain.
A plasmid containing fae under the expression of its own putative promoter, pCM139, was used to complement the fae::kan mutant strain. pCM139 was constructed as follows. The 0.8-kb region containing fae and its putative promoter region was amplified by PCR using the primers CM-Pfaef, 5'-GGATCCTGAGCCTTGGTCCAG-3', and CM-4010r, 5'-TGACTGCCTCCGATCTAAG-3'. The resulting PCR product was cloned into pCR2.1 TOPO TA (Invitrogen) to generate pCM138. This region was then excised with BamHI and SphI and inserted into a broad-host-range cloning vector recently developed for M. extorquens AM1, pCM62 (C. J. Marx and M. E. Lidstrom, unpublished data), to create pCM139.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Fae activity in cell extracts. Cell extracts of M. extorquens AM1 grown in the presence of methanol were found to accelerate the reaction of formaldehyde and H4MPT to methylene H4MPT. The activity catalyzing the acceleration of this reaction is designated Fae. A unit of activity was defined as the formation of 1 µmol of methylene H4MPT from H4MPT and formaldehyde per min minus the spontaneous rate without enzyme added. Under standard assay conditions at pH 8.0, the Fae activity of cell extracts from cells grown in the presence of methanol was found to be 1.4 U/mg. Cell extracts of cells grown on succinate exhibited an activity of only 0.3 U/mg, showing that the activity is induced upon growth on methanol.
No acceleration of the spontaneous reaction of formaldehyde and H4F was observed at various pH values and buffers in cell extracts of M. extorquens AM1. Also, no formaldehyde-H4F condensing activity could be observed with purified Fae.Localization and purification of Fae.
The Fae activity was
recovered in the soluble fraction of the cell extract; membrane
fractions did not show activity. After three chromatographic steps,
preparations contained only one polypeptide, with an apparent molecular
mass of 18 kDa, as revealed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (Fig. 2) and a
specific activity of about 20 U/mg. Purification was about 14-fold with
a yield of about 23% (Table 1),
indicating that the protein is present in relatively large amounts in
the cell, 2% or more.
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Molecular and catalytic properties of Fae. The apparent molecular mass of Fae was determined by gel filtration on Superdex 200 to be about 60 kDa. Since the subunit molecular mass of Fae was found to be 18 kDa, this finding suggests that Fae has a homotrimeric structure. The UV-visible spectrum of the enzyme was that of a protein lacking a chromophoric prosthetic group.
The spontaneous reaction of formaldehyde with H4MPT and the enzyme-catalyzed reaction exhibited different pH optima (Fig. 3). Whereas the spontaneous reaction proceeded faster in slightly acidic conditions in potassium phosphate buffer, the enzymatic reactions had a more alkaline pH optimum. When a 50 mM Tricine-KOH buffer system was used at pH 8.0, the presence of 30 mM MgCl2 or MgSO4 stimulated the activity about eightfold (Ka = 4 mM). Higher MgCl2 or MgSO4 concentrations did not result in higher reaction rates.
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Identification of the gene encoding Fae and similarities to putative proteins from methanogenic archaea. The N-terminal 40 amino acids of Fae from M. extorquens AM1 were determined by Edman degradation: AKITKVQVGEALVGDGNEVAHIDLIIGPXGSPAETAFXNG. The sequence and predicted molecular mass of 18 kDa were homologous with the gene product previously designated orf18 (7), which is now named fae.
The gene encoding Fae is located within a cluster of genes for proteins with sequence similarities to proteins from methanogenic and sulfate-reducing archaea (7). This cluster contains both the genes for the H4MPT-methanofuran-dependent enzymes involved in the further oxidation of methylene H4MPT to CO2 (Fig. 1) and open reading frames of unknown function (7, 27). Fae exhibits amino acid sequence identities of 50, 49, and 25%, respectively, to the N-terminal portion of putative proteins of unknown function deduced from the genome sequences of Archaeoglobus fulgidus (Orf1305), M. thermoautotrophicumH (Orf1474), and Methanococcus
jannaschii (Orf1447) (4, 21, 31). We could not,
however, detect a formaldehyde-H4MPT condensing activity in
cell extracts of M. thermoautotrophicum Marburg and M. barkeri Fusaro.
Construction of Fae-minus mutants and phenotypical analysis. In order to assess the physiological importance of Fae activity, a mutant strain with an interrupted fae::kan allele was generated by allelic exchange. The fae::kan mutant strain CM115.1 lacked detectable Fae activity and was unable to grow on methanol but exhibited wild-type growth on succinate (data not shown). Furthermore, growth of the fae::kan mutant strain on succinate-containing solid media was inhibited by the addition of methanol or formaldehyde at MICs of 50 and 200 µM, respectively. These results suggest that the Fae-H4MPT-dependent pathway is the primary formaldehyde detoxification system during growth on both one-carbon and multicarbon compounds. Introduction of pCM139, a plasmid bearing only fae with its putative promoter region, restored wild-type growth to CM115.1, thus confirming that the phenotype is not due to a polar effect on downstream genes.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have shown that M. extorquens AM1 contains a protein, Fae, which catalyzes the condensation of formaldehyde and H4MPT to methylene H4MPT (Fig. 1). From the rates shown in Fig. 3, it can be calculated that the spontaneous rate of formaldehyde condensation with H4MPT at physiological pH is not sufficient to accommodate the formaldehyde production rate but that the consumption rate with Fae would be sufficient. Accordingly, a fae knockout strain was found to be unable to grow on methanol. In addition, the mutant was shown to be formaldehyde sensitive, suggesting an involvement in formaldehyde detoxification of M. extorquens AM1. In this case, it is likely that Fae acts together with the other H4MPT-dependent enzymes to detoxify formaldehyde. The fae gene is indeed clustered with a group of other archaeon-like genes that encode the remainder of the H4MPT-dependent formaldehyde oxidation pathway. It has been previously suggested that this pathway was acquired by horizontal transfer from an ancestral archaeon (7, 34). Our results suggest that Fae may also have been acquired in this way, possibly in the same transfer event.
We did not find a formaldehyde-H4F condensing activity in cell extracts of M. extorquens AM1, nor did purified Fae show this activity. The condensation of formaldehyde with H4F occurs also spontaneously in vitro. The rates are comparable to the rates of condensation of formaldehyde and H4MPT and might be sufficient for methylene H4F formation, which is required for assimilation by the serine cycle and most likely purine and formylmethionine tRNA biosynthesis. The formation of methylene H4F might simply be regulated by the availability of free H4F and formaldehyde. Our hypothesis that the main flux of formaldehyde proceeds via the H4MPT- and not the H4F-dependent pathway is in agreement with an enzyme which accelerates the condensation of formaldehyde with H4MPT.
The only orthologs of Fae are found in methanogenic and
sulfate-reducing archaea (Fig. 5). UP to
now, the function of these orthologs has been unknown. Their occurrence
raises the question as to why methanogens would contain a
formaldehyde-H4MPT condensing activity. Formaldehyde
is not thought to be a central metabolite of methanogenesis, although
formaldehyde can be used as a methanogenic substrate in cell extracts
and cell suspensions of methanogenic archaea, albeit only very slowly
(14, 24, 26). We could not detect a
formaldehyde-H4MPT condensing activity in cell extracts of
the methanogenic archaea tested. It is possible, however, that the Fae
ortholog in these organisms either is inactive or has activity with a
substrate other than H4MPT, formaldehyde, or both. Interestingly, the Fae orthologs in the methanogenic and
sulfate-reducing archaea are fused to a second, C-terminal domain that
shows identity to 3-hexulose-6-phosphate synthase (HPS, encoded by
rmpA) (Fig. 5B) from Methylomonas aminofaciens
77a (35) (Fig. 5B). HPS is the initial
formaldehyde-utilizing enzyme found in methylotrophic bacteria that use
the ribulose monophosphate pathway for formaldehyde assimilation
(35) (Fig. 5B). HPS catalyzes the condensation of
formaldehyde with D-ribulose-5-phosphate to generate
D-arabino-3-hexulose-6-phosphate, which in ribulose
monophosphate pathway methylotrophs is then isomerized to
fructose-6-phosphate by 6-phospho-4-hexuloisomerase (PHI, encoded by
rmpB) (23, 29). Curiously, HPS and PHI orthologs can be found in a number of genomes of nonmethylotrophic bacteria and
nonmethanogenic archaea (Fig. 5B) (19, 28, 37). The HPS and
PHI orthologs were recently cloned and purified from Bacillus subtilis, where they apparently function as a formaldehyde
detoxification system (37). In the hyperthermophilic archaea
Pyrococcus horikoshii and Pyrococcus abyssi, the
HPS and PHI orthologs are fused into a single polypeptide
(19). The in vivo function of these fusion proteins of
fae and rmpA as well as rmpA and
rmpB remains to be demonstrated in archaea and may provide
new insights into formaldehyde conversion in archaea. The widespread
occurrence of orthologs to this group of genes encoding formaldehyde
utilization enzymes suggests that formaldehyde may play an unknown but
important cellular role in a broad group of prokaryotes.
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ACKNOWLEDGMENTS |
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This work was supported by the Max Planck Society and by a grant from the NIH (GM36296) to M.E.L.
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FOOTNOTES |
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* Corresponding author. Mailing address: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany. Phone: (49) 6421-178333. Fax: (49) 6421-178209. E-mail: vorholt{at}mailer.uni-marburg.de.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, activity in
the absence of enzyme (spontaneous rate); 
, activity in the presence
of enzyme; 
, activity in the presence of enzyme corrected for the
spontaneous rate.
