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Journal of Bacteriology, December 2000, p. 6645-6650, Vol. 182, No. 23
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
Julia A.
Vorholt,1,*
Christopher J.
Marx,2
Mary E.
Lidstrom,2,3 and
Rudolf K.
Thauer1
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
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ABSTRACT |
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
-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.
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INTRODUCTION |
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
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.
The catabolism of C1 units is best studied in
Methylobacterium extorquens AM1. The -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:
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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.
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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 |
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.
Activity of purified Fae was also measured in a coupled assay at 340 nm
together with purified methylene H
4MPT dehydrogenase
(MtdA)
(
33), which catalyzes the dehydrogenation of methylene
H
4MPT. Reaction rates of Fae in the coupled assay were
calculated
indirectly through differences in MtdA
activity.
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.
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RESULTS |
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
H
4F was observed at various pH values and buffers in cell
extracts of
M. extorquens AM1. Also, no
formaldehyde-H
4F 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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FIG. 2.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis of fractions of Fae upon purification from
M. extorquens AM1. Lane M, molecular mass standards
(Amersham-Pharmacia Biotech); lane 1, 20 µg of cell extract protein;
lane 2, 8 µg of protein after DEAE-Sephacel; lane 3, 6 µg of
protein after Q-Sepharose; lane 4, 4 µg of protein after
hydroxyapatite.
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The activity of purified Fae could also be measured in a coupled assay
together with purified methylene H
4MPT dehydrogenase
(MtdA), which catalyzes the dehydrogenation of methylene
H
4MPT
(
33). Reaction rates of Fae were
calculated indirectly through
differences in MtdA activity and
corresponded very well with the
activity of Fae determined directly at
250 nm. This experiment
confirms that the reaction products formed by
spontaneous reaction
of formaldehyde and H
4MPT as well as
the reaction catalyzed by
Fae yield the same product, namely,
N5,
N10-methylene
H
4MPT. Furthermore, the experiment supports the assumption
that in vivo the formation of methylene H
4MPT catalyzed by
Fae
is followed by its dehydrogenation catalyzed by methylene
H
4MPT
dehydrogenase (MtdA or MtdB), which shows high
activities of several
units per mg in cell extracts (Fig.
1) (
12,
33).
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 H
4MPT 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 MgCl
2 or MgSO
4 stimulated the activity about
eightfold (
Ka = 4 mM).
Higher MgCl
2
or MgSO
4 concentrations did not result in higher
reaction
rates.
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FIG. 3.
pH optimum of the spontaneous reaction of formaldehyde
with H4MPT and of the reaction catalyzed by Fae. The assays
contained 120 mM potassium phosphate, 40 µM H4MPT, 1.6 mM
CH2O, and 5 µg of purified Fae when indicated. Assays
were performed at the pH value indicated at 30°C.  , 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.
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Fae activity depending on the formaldehyde concentration followed
Michaelis-Menten kinetics and showed a high affinity of
Fae for
formaldehyde. Reciprocal plots indicated an apparent
Km for formaldehyde of 0.2 mM (Fig.
4A). For the determination of
the
Km value of H
4MPT in the assay, only
up to 60 µM H
4MPT could
be used because of technical
reasons. Reciprocal plots indicated
an apparent
Km value of about 1 mM H
4MPT (Fig.
4B). The relatively
high apparent
Km for
H
4MPT may be due to the fact that H
4MPT from
Methanobacterium thermoautotrophicum rather than
dephospho-H
4MPT
from
M. extorquens AM1
(
33) was used in the assays.
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FIG. 4.
Kinetics of Fae with respect to different formaldehyde
and H4MPT concentrations. The assay mixtures contained 50 mM Tricine-KOH (pH 8.0), 30 mM MgCl2, and 1.5 µg of
purified Fae; the H4MPT concentration was 50 µM (A) or as
indicated (B), and the CH2O concentration was as indicated
(A) or 1.6 mM (B).
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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 H
4MPT-methanofuran-dependent enzymes involved
in the further oxidation of methylene H
4MPT to
CO
2 (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. thermoautotrophicum H (Orf1474), and
Methanococcus
jannaschii (Orf1447) (
4,
21,
31). We could not,
however, detect a
formaldehyde-H
4MPT 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 |
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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FIG. 5.
(A) Alignment of the amino acid sequence of Fae from
M. extorquens AM1 and putative proteins from the complete
genomes of the sulfate-reducing archaeon A. fulgidus
(AF1305) (21) and the methanogenic archaeon M. thermoautotrophicum H (Mth1474) (31). (B) Sequence
analysis of orthologs of Fae from M. extorquens AM1. Fae
from M. extorquens AM1 shows sequence identity to the
N-terminal domain of putative proteins from A. fulgidus,
M. thermoautotrophicum H, and M. jannaschii
(4, 21, 31). The C-terminal domains of these archaeal
proteins themselves show sequence identity to 3-hexulose-6-phosphate
synthase (HPS; RmpA) from M. aminofaciens (35). A
homologue of RmpA is linked to the 6-phospho-4-hexuloisomerase (PHI;
RmpB) homologue from M. aminofaciens (29) in
Pyrococcus species (19).
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ACKNOWLEDGMENTS |
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 |
*
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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Journal of Bacteriology, December 2000, p. 6645-6650, Vol. 182, No. 23
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
