From the
The archaeon Pyrococcus furiosus grows optimally at 100
°C by the fermentation of carbohydrates to yield acetate,
CO
Hyperthermophiles are a recently discovered group of
microorganisms that have the remarkable property of growing at
temperatures of 90 °C and above
(1, 2) . Almost all
are classified as Archaea (formerly Archaebacteria, Ref. 3), and the
majority are strictly anaerobic heterotrophs that reduce elemental
sulfur (S°) to H
From a very recent study, however, which
used NMR and enzymatic analyses to investigate the metabolism of
[
No activity was detected in the routine
assay of GAPOR when benzyl viologen was replaced by NADP or NAD (each 5
m
M), but P. furiosus ferredoxin did function as an
electron carrier for the enzyme. The K
There was no detectable reduction of benzyl viologen
when GAP was replaced in the routine assay of GAPOR at 70 °C by
acetaldehyde, crotonaldehyde, formaldehyde, propionaldehyde,
butyraldehyde, benzaldehyde, phenylacetaldehyde, glyceraldehyde
(measured at 40 °C, see Ref. 9), glucose, glucose 6-phosphate,
dihydroxyacetone phosphate, glycerol 3-phosphate, or glyoxalate, each
at a concentration of 1.0 or 10 m
M. Similarly, there was no
significant effect on the activity of GAPOR using GAP as the substrate
when glucose, glucose 6-phosphate, dihydroxyacetone phosphate, glycerol
3-phosphate, or phosphoglycolate, each at a concentration of 10
m
M, were included in the routine assay. The presence of
crotonaldehyde (10 m
M) or formaldehyde (10 m
M)
decreased GAPOR activity by approximately 25%, whereas acetyl
phosphate, 3-phosphoglycerate, or 2,3-bisphosphoglycerate (each 10
m
M) stimulated GAPOR activity by between 58 and 93%.
When
sodium dithionite was added to GAPOR (1.0 mg/ml in 50 m
M
Tris/HCl, pH 8.0) to a final concentration of 3.0 m
M, less
than 50% of the GAP oxidation activity was observed. When the
dithionite was then removed from the enzyme by gel filtration,
approximately 70% of the original specific activity was recovered. The
substrate specificity of GAPOR was unaffected when the enzyme was
prepared with and without dithionite. The substrate specificities of
AOR and FOR from P. furiosus (which are purified in the
presence of sodium dithionite) were also unaffected when sodium
dithionite was removed (in particular, GAP was not a substrate for
either enzyme). GAPOR was routinely assayed in the absence of phosphate
ions and its activity was unaffected by the presence of 5 m
M
K
, and H
. Cell-free extracts contain very low
activity of the glycolytic enzyme, glyceraldehyde-3-phosphate
dehydrogenase, but extremely high activity of
glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). GAPOR was
purified under strictly anaerobic conditions. It is a monomeric,
O
-sensitive protein of M
63,000
which contains pterin and approximately 1 tungsten and 6 iron atoms per
molecule. The enzyme oxidized glyceraldehyde-3-phosphate
( K
28 µ
M) to
3-phosphoglycerate and reduced P. furiosus ferredoxin
( K
6 µ
M), but it did not
oxidize formaldehyde, acetaldehyde, glyceraldehyde, benzaldehyde,
glucose, glucose 6-phosphate, or glyoxylate, nor did it use NAD(P) as
an electron acceptor. It is proposed that GAPOR has a glycolytic role
and functions in place of glyceraldehyde-3-phosphate dehydrogenase and
possibly phosphoglycerate kinase.
S. The best studied of these is
Pyrococcus furiosus ( T
100 °C),
which also grows well in the absence of S°
(4) . This
organism utilizes both complex (starch, glycogen) and simple (maltose,
cellobiose) sugars as a carbon source but monosaccharides (glucose,
fructose) do not support growth. The fermentation products are acetate,
H
and CO
. Alanine is also produced at high
partial pressures of H
(5) . The growth of P.
furiosus is dependent upon tungsten
(6) , an element seldom
used in biological systems
(7) , and two tungsten-containing
enzymes, aldehyde ferredoxin (Fd)
(
)
oxidoreductase (AOR, Refs. 8 and 9) and formaldehyde Fd
oxidoreductase (FOR, Refs. 10 and 11) have been purified. AOR oxidizes
a range of aliphatic and aromatic aldehydes in vitro (9) , whereas FOR utilizes only C1-C3 aldehydes. FOR
is thought to function in peptide catabolism
(11) , whereas it
was hypothesized that AOR catalyzed glyceraldehyde oxidation
(9) as part of an unusual nonphosphorylated Entner-Doudoroff
(ED) pathway. Most of the other enzymes of this ED pathway were
subsequently demonstrated in cell extracts, albeit with very low
activities
(12) .
C]glucose by cell suspensions of P.
furiosus, it was concluded that this occurs via an unusual
Embden-Meyerhof (EM) pathway which contains ADP- (AMP-forming) rather
than ATP-dependent hexose(-phosphate) kinases
(13) . A similar
study by others
(14) also concluded that an EM type of pathway
was present in P. furiosus, although a version of the ED
pathway was also proposed. It is not clear, however, how an EM-type
pathway could function in this organism, because cell extracts contain
very low activities of two key glycolytic enzymes,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate
kinase (PGK)
(9, 12, 13, 15) . In fact,
these enzymes are thought to function in gluconeogenesis when P.
furiosus is grown on pyruvate
(15) . We show here that
P. furiosus contains high activity of a new type of enzyme,
glyceraldehyde-3-phosphate Fd oxidoreductase (GAPOR). This is proposed
to function in place of GAPDH and possibly PGK in the novel EM-type
pathway. Moreover, GAPOR represents the third tungsten-containing
enzyme to be purified from P. furiosus.
Growth of the Organism and Enzyme
Purification
P. furiosus (DSM 3638) was grown in the
absence of S° at 90 °C in a stainless steel 600-liter fermentor
using maltose as the carbon source
(6) . GAPOR was purified from
100 g (wet weight) of frozen cells under strictly anaerobic conditions
at 23 °C. In all chromatography steps, the columns were washed with
at least 1 column volume of buffer before the absorbed proteins were
eluted. The techniques and procedures were the same as those used to
purify P. furiosus AOR
(9) , up to and including the
first chromatography step, except that sodium dithionite and glycerol
were omitted from all buffers. GAPOR activity eluted from the first
column (5.0 10 cm) of DEAE-Sepharose Fast Flow (Pharmacia
Biotech Inc.) at 0.12
M NaCl in buffer A (50 m
M
Tris/HCl, pH 8.0, containing 2 m
M dithiothreitol). Fractions
containing GAPOR activity (762 mg, 6,200 units) were applied to a
column (2.5
10 cm) of hydroxyapatite (Bio-Rad) previously
equilibrated with buffer A at 2.0 ml/min. Absorbed proteins were eluted
with a linear gradient (1,000 ml) from 0 to 200 m
M
K
phosphate in buffer A at 2 ml/min. GAPOR activity
eluted as 10 m
M K
phosphate was applied.
Active fractions (310 mg, 5,250 units) were concentrated by
ultrafiltration with an Amicon-type PM-30 membrane and applied to a
column (1.6
60 cm) of Superdex 200 (Pharmacia) equilibrated
with buffer A containing 200 m
M KCl at 3 ml/min.
GAPOR-containing fractions (50 mg, 4,250 units) were concentrated and
washed with buffer A and then applied to a column (1.2
10 cm)
of Blue Sepharose (Pharmacia) equilibrated with buffer A. Absorbed
proteins were eluted with a linear gradient (120 ml) from 0 to 300
m
M KCl. GAPOR activity eluted as 180 m
M KCl was
applied. Those fractions containing homogenous GAPOR as judged by
electrophoresis (11 mg, 1,540 units) were concentrated and stored as
pellets in liquid N
Enzyme Assays and Other Methods
The activity
of GAPOR was determined at 70 °C by the reduction of benzyl
viologen (3.0 m
M) measured at 580 nm (molar absorbance 7,800
Mcm
) in 100 m
M
EPPS buffer, pH 8.4 (see Ref. 9), using glyceraldehyde 3-phosphate
(GAP, 0.4 m
M) as the substrate. GAP was determined to have a
half-life at 70 °C of 2 min under routine assay conditions (as
determined using rabbit muscle GAPDH (Sigma) at 23 °C). Therefore,
it was added to start the reaction in all assays of GAPOR, and
activities were calculated from the first 10 s of benzyl viologen
reduction. The activities of AOR
(9) and FOR
(11) were
determined at 80 °C by the oxidation of crotonaldehyde and
formaldehyde, respectively. GAPDH activity was measured at 70 °C by
following the reduction of NAD at 340 nm
(16) . For all enzymes,
1 unit of activity is defined as 1 µmol of substrate oxidized per
min. Concentrations of 3-phosphoglycerate (0-2.0 m
M)
were estimated using chicken liver 3-phosphoglycerate dehydrogenase
(Sigma) at 23 °C. Molecular weight estimation by gel filtration
(Superdex 200) and SDS-electrophoresis, protein analyses, plasma
emission and fluorescence spectroscopy, colorimetric iron
determination, and N-terminal amino acid sequence analysis were all
carried out as described previously
(9, 17) . The pterin
was extracted from purified GAPOR by I
/KI treatment
(9) . NADP reduction using GAP (0.4 m
M) as the
substrate was determined at 70 °C using a system containing GAPOR
(1.5 mg/ml), P. furiosus ferredoxin (0.38 mg/ml), P.
furiosus ferredoxin NADP oxidoreductase (0.1 mg/ml), and NADP (0.4
m
M) under previously described conditions
(18) .
Purification of GAPOR
Anaerobically prepared
cytoplasmic extracts of three different batches of P. furiosus cells grown on maltose contained high GAPOR activity
(4.7-8.2 units/mg with benzyl viologen as the electron acceptor,
1.2-2.6 units/mg using P. furiosus ferredoxin as the
electron acceptor; see below), but the GAPDH activity was very low
(0.07-0.09 unit/mg using the same cell extracts). Under aerobic
conditions, cell extracts lost about 50% of GAPOR activity after 12 h
at 23 °C, but no activity was lost if the extract was maintained
under argon. The enzyme was therefore purified under anaerobic
conditions but in the absence of the O-scavenger, sodium
dithionite, which is an inhibitor (see below). Using DEAE-Sepharose
Fast Flow chromatography, GAPOR and FOR activities (eluting at 0.12
M NaCl) were separated from related activities in the
cell-free extract such as AOR (0.18
M NaCl) and GAPDH (0.24
M NaCl). GAPOR also co-eluted with FOR from a hydroxyapatite
column but the two were easily separated by Superdex S-200
chromatography (data not shown). Two separate purification procedures
each yielded about 11 mg of purified GAPOR with a specific activity of
about 140 units/mg. A 30-fold purification was achieved compared with
the cell-free cytoplasmic extract.
Physical Properties of GAPOR
As shown in Fig. 1,
purified GAPOR prepared by heating at 100 °C for 10 min with SDS
(1.0%, w/v) gave rise to two protein bands after SDS-electrophoresis
(10%, w/v, acrylamide) which corresponded to approximate
Mvalues of 44,000 and 63,000. If the sample was
not heated, only the M
44,000 band was observed
(Fig. 1), suggesting that this form of the enzyme was not
completely unfolded. Incomplete denaturation by SDS treatment has also
been reported for some other enzymes from P. furiosus (see
Refs. 2 and 6). However, if purified GAPOR was precipitated with
trichloroacetic acid (10%, w/v) prior to the heat treatment step, only
the M
63,000 band was evident (Fig. 1). This
band was therefore assumed to represent the fully denatured protein.
Both protein bands gave the same N-terminal sequence (see below),
confirming that they are different denatured forms of the same protein.
Analysis of GAPOR by gel filtration (in the presence of 1.0
M
NaCl) gave an M
value of 60,000 ± 5,000.
Together with the electrophoretic data, these results suggest that the
enzyme is a monomeric protein of M
approximately
63,000. The presence of a single subunit was confirmed by N-terminal
amino acid sequence analysis of a solution of GAPOR which gave rise to
a single sequence (M K F S V L K L D V G K R E V E A Q E I E R E D I F
G V V D Y G I M R H N E). This showed no homology to the N-terminal
sequences of either AOR or FOR from P. furiosus (11) or to GAPDH from Pyrococcus woesei (16) .
Figure 1:
Analysis of purified P.
furiosus GAPOR by SDS-electrophoresis. Samples of GAPOR (2.0
mg/ml) were incubated with an equal volume of SDS (1%, w/v) prior to
electrophoresis on a 10% (w/v) acrylamide gel. The samples were:
standard proteins -galactosidase (116,000, top),
phosphorylase b (97,400), bovine serum albumin (67,000), and
ovalbumin (45,000, bottom) ( lane 1); GAPOR
precipitated with trichloroacetic acid and then heated with SDS at 100
°C for 10 min ( lane 2); GAPOR treated with SDS at 25
°C ( lane 3); GAPOR heated with SDS at 100 °C for 10
min ( lane 4).
GAPOR contained 0.85 ± 0.12 tungsten and 5.0 ± 1.6
iron g atoms/mol when analyzed by plasma emission spectroscopy and 5.9
± 0.4 iron g atoms/mol by colorimetric analysis (each value is
the average of two determinations using two different enzyme
preparations). The enzyme as isolated gave a broad visible absorption
band around 420 nm, and this decreased by nearly 50% upon addition of
sodium dithionite (data not shown), consistent with the presence of
iron-sulfur chromophores. The spectra were very similar to those
obtained with P. furiosus AOR, which included a broad feature
near 550 nm in the oxidized form (see Fig. 1of Ref. 8). Material
extracted from GAPOR after acid denaturation and oxidation with
I/KI gave fluorescence spectra that were also very similar
to those obtained after treating P. furiosus AOR in a similar
manner (see Fig. 1of Ref. 9). These spectra are characteristic
of a pterin-type cofactor, suggesting that the tungsten in GAPOR is
present as a tungstopterin center, as it is in AOR and also FOR
(10) . The N-terminal sequence of GAPOR showed no homology to
the subunits of the two other types of tungsten-containing enzymes that
are known, carboxylic acid reductase
(19) and
formylmethanofuran dehydrogenase
(20) . Notably, the
tungstoenzymes AOR, FOR, and carboxylic acid reductase share a high
degree of N-terminal homology
(11) .
Catalytic Properties of GAPOR
GAPOR was purified
by its ability to oxidize GAP at 70 °C using benzyl viologen as the
electron acceptor. Since GAP had a half-life under assay conditions of
2 min, it was important to establish that a degradation product of GAP
was not serving as a substrate for GAPOR. This was substantiated by the
following. When GAP was completely degraded by a 5-min incubation at 80
°C under assay conditions (there was no reduction of benzyl
viologen during this process); upon the addition of GAPOR, the
reduction of benzyl viologen was observed only when freshly prepared
GAP was subsequently added. These results also indicated that the
thermal degradation products of GAP did not inhibit GAPOR. Using GAP at
concentrations of 0.01-0.50 m
M (benzyl viologen, 3
m
M), the Kand V
values obtained from a linear double-reciprocal plot were 30
µ
M and 350 units/mg, respectively. There was significant
inhibition of GAPOR using GAP concentrations above 0.5 m
M.
Using benzyl viologen at concentrations of 0.01-5.0 m
M
(GAP, 0.40 m
M), the K
and
V
values were 0.43 m
M and 310 units/mg,
respectively. To investigate whether 3-phosphoglycerate was the product
of GAPOR activity, the reaction was allowed to proceed under standard
assay conditions at 70 °C for 2 min. The reaction mixture was then
rapidly cooled to 4 °C, and the concentration of 3-phosphoglycerate
was determined enzymatically to be 95 µ
M. This compares
with a calculated value of 110 µ
M, based on the initial
rate of benzyl viologen reduction. 3-Phosphoglycerate could not be
detected if the same experiment was performed in the absence of GAPOR,
which also shows that 3-phosphoglycerate was not a significant thermal
degradation product of GAP.
and V
values calculated from a linear
Lineweaver-Burk plot were approximately 6 µ
M and 90
units/mg, respectively (using 0.1-50 µ
M ferredoxin
and 0.40 m
M GAP). Using GAP at concentrations of
0.01-0.50 m
M (ferredoxin, 100 µ
M), the
K
and V
values
obtained were 28 µ
M and 78 units/mg, respectively. The
high activity obtained with ferredoxin as the electron carrier and the
high affinity of GAPOR for both GAP and ferredoxin suggest that these
are the physiological substrates. Since NADPH rather than reduced
ferredoxin is the physiological electron donor to the
H
-evolving hydrogenase of P. furiosus (18) , it was of interest to determine if GAP oxidation
could be coupled to NADP reduction. A combination of GAPOR, ferredoxin,
and P. furiosus ferredoxin NADP oxidoreductase (FNOR, Ref. 17)
reduced NADP at a rate of 88 µmol/min/mg (of FNOR) at 70 °C,
suggesting that in vivo, reductant from the GAPOR reaction
could be readily converted to NADPH and disposed of via hydrogenase as
H
.
phosphate in the assay medium. However, K
phosphate at a concentration of 200 m
M stimulated GAPOR
activity by approximately 3.5-fold, and Na
arsenate
(200 m
M) had a similar effect. By analogy with GAPDH, which
generates 1,3-bisphosphoglycerate from GAP, these results suggest that
GAPOR in the presence of phosphate might also produce
1,3-bisphosphoglycerate. We could not detect 1,3-bisphosphoglycerate
(using rabbit muscle GAPDH at 25 °C) as a product of the GAPOR
reaction when the standard assay was carried out for 2 min at 70 °C
in the presence of 200 m
M K
phosphate,
whereas the concentration of 3-phosphoglycerate produced under these
conditions was determined enzymatically to be 285 µ
M,
which was in good agreement with the calculated value (350
µ
M, based on the initial rate of benzyl viologen
reduction). This result is not conclusive, however, because
1,3-bisphosphoglycerate is very unstable and rapidly hydrolyzes to
3-phosphoglycerate and inorganic phosphate (even at 25 °C, Ref.
21), and so it may have rapidly degraded under assay conditions. On the
other hand, GAPOR activity was also stimulated about 3.5-fold by the
presence of potassium chloride, sodium citrate, or sodium sulfate (each
200 m
M), showing that there is general enhancement of GAPOR
activity by high ionic strength and particularly by polyanions,
including phosphate.
Physiological Function of GAPOR
The oxidation of
GAP to 1,3-bisphosphoglycerate and its conversion to 3-phosphoglycerate
is usually accomplished by a combination of the glycolytic enzymes,
GAPDH and PGK. The activities of these two enzymes in maltose-grown
P. furiosus are both very low when assayed in the glycolytic
direction (0.06 units/mg at 90 °C, Refs. 13 and 15), yet it has
been shown that an EM-type glycolytic pathway is the predominant route
for glucose oxidation in this organism
(13) . It therefore seems
reasonable to suggest that GAPOR is a glycolytic enzyme and functions
in place of GAPDH and PGK. As shown in Fig. 2, it is proposed
that GAPOR produces 3-phosphoglycerate and that the reductant is
disposed of as H
via NADPH (see Ref. 18). An attractive
feature of this pathway is that all of the enzymes involved
exhibit-specific activities of greater than 0.5 units/mg at 70-90
°C using cell-free extracts of maltose-grown P. furiosus (2, 6, 12, 13, 15, 17) .
Moreover, assuming that the ADP required for hexose(-phosphate)
activation is readily available from anabolic processes
(13) ,
the proposed pathway yields 4 mol of ATP/mol of glucose oxidized to
acetate, which is in accordance with growth yield data (see Refs. 12,
13, and 22). We cannot exclude the possibility that in vivo GAPOR produces 1,3-bisphosphoglycerate, but this would seem
unlikely since ( a) if 1,3-bisphosphoglycerate was used by PGK
to generate ATP, the overall ATP yield (6 ATP/glucose) would exceed
that determined experimentally (see Refs. 12 and 22), and (b) the
activities of both PGK and GAPDH in P. furiosus increase
dramatically (up to 10-fold) in pyruvate-grown cells, suggesting they
function primarily in gluconeogenesis
(15) .
Figure 2:
Proposed role of GAPOR in the conversion
of glucose to acetate in P. furiosus. Enzymes that have been
purified are shown in bold and underlined, unusual enzymes are
indicated, and dotted lines indicate electron transfer
reactions. Data were taken from Refs. 2, 13, 17, and
18.
The EM-type
pathway shown in Fig. 2also satisfies the requirement of ATP
production during glucose oxidation to pyruvate
(22) , which
does not occur in the ED-type pathway proposed for P. furiosus (12) . Indeed, the operation of this ED pathway is also
questionable in light of the very low activities of some of the enzymes
involved
(12) . A notable exception in this regard is AOR
(9) , which perhaps necessitates a reevaluation of its
physiological function. In any event, we have demonstrated that P.
furiosus GAPOR is distinct from AOR and also from FOR, the other
tungstoenzymes in this organism. GAPOR is also unrelated to GAPDH or to
any other known tungstoprotein. The enzyme is present at a relatively
high cellular concentration (about 3% of the total protein), consistent
both with its function in the primary pathway of sugar metabolism and
with the dependence of cell growth upon tungsten
(6, 8) . Although the product of the GAPOR reaction
in vivo remains undetermined, the pathway shown in
Fig. 2
will serve as a guide for future metabolic and
enzymological studies.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.