Department of Biochemistry and Molecular
Biology and Center for Metalloenzyme Studies, University of
Georgia, Athens, Georgia 30602,1 and
Department of Genetics, University of Utah, Salt Lake City,
Utah 841122
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INTRODUCTION |
In the last decade, several species
of anaerobic sulfur-dependent heterotrophic hyperthermophiles have been
isolated from solfataric fields and submarine hydrothermal
systems (5, 46). These organisms exhibit optimal
growth at temperatures of 90°C or above, and most of them belong to
the domain Archaea rather than Bacteria
(50). With the exception of the methanogenic species, many
of the hyperthermophiles are able to utilize peptides as their sole
carbon source, although a few saccharolytic species are also known. One
of the most extensively studied of these organisms is the archaeon
Pyrococcus furiosus (optimum growth temperature, 100°C
[14]). This anaerobic heterotroph grows on peptides
(casein, peptone, yeast extract) and utilizes both simple
(maltose, cellobiose) and complex (starch, glycogen) sugars. It
metabolizes carbohydrates by an unusual fermentation-type pathway
whose products are acetate, CO2, H2, and
alanine (26, 27). Although the growth of many of the
hyperthermophilic archaea is dependent upon elemental sulfur (S0), which is reduced to H2S, P. furiosus is one of the few archaea that can grow well in the
absence of S0 (14, 44).
The growth of P. furiosus is dependent upon tungsten
(8), an element rarely used in biological systems (24,
30). Two tungsten-containing, aldehyde-oxidizing enzymes,
aldehyde ferredoxin oxidoreductase (AOR [36]) and
glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR
[38]) have been previously purified from this
organism. AOR has a broad substrate specificity but is most active with aldehydes derived from amino acids (via transamination and
decarboxylation [21]). AOR is thought to play a
key role in peptide fermentation by oxidizing aldehydes generated by
the four types of 2-keto acid oxidoreductases present in this organism (1, 22, 35). In contrast, GAPOR uses only
glyceraldehyde-3-phosphate as a substrate and it functions in the
unusual glycolytic pathway that is present in P. furiosus, replacing the expected glyceraldehyde-3-phosphate dehydrogenase (38).
The hyperthermophilic archaeon Thermococcus litoralis has
been reported to contain another type of tungsten-containing,
aldehyde-oxidizing enzyme, termed formaldehyde ferredoxin
oxidoreductase (FOR [37]). This organism, like
P. furiosus, ferments peptides and certain sugars and
requires S0 for optimal growth (7, 39), although
T. litoralis shows essentially no similarity (3%) with
P. furiosus at the DNA level (39). FOR was
reported to oxidize only small (C1 to C3)
aliphatic aldehydes (37). However, dramatic losses of FOR
activity occurred during purification from T. litoralis
(>90% after two chromatography steps), leading to a virtually
inactive enzyme (37). Herein we show that P. furiosus also contains FOR and report on its biochemical and
molecular properties, including the gene-derived amino acid sequence.
This enzyme also undergoes significant inactivation during
purification, but the results presented herein indicate that this is
due to the loss of sulfide. The substrate specificity of FOR (both
purified and sulfide-activated FOR) has been evaluated, and although
its function in vivo is still unclear, the enzyme shows a high
catalytic efficiency with aliphatic dialdehydes.
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MATERIALS AND METHODS |
Growth of the organism and enzyme purification.
P.
furiosus (DSM 3638) was grown in the absence of S0 at
90°C in a stainless steel fermentor with maltose as the carbon source as previously described (8). FOR was routinely purified from frozen cells (500 g [wet weight]) under strictly anaerobic conditions at 23°C. The buffer used throughout the purification was 50 mM Tris-HCl, pH 8.0, containing 2 mM dithiothreitol (DTT). Where indicated
(see Results), 10% glycerol (vol/vol) and/or 2 mM sodium dithionite
were also present. The techniques and procedures were the same as those
used to purify AOR from P. furiosus, up to the first
chromatography step (36). In a typical purification, the cell extract prepared from up to 500 g (wet weight) of frozen cells was loaded onto a column (10 by 14 cm) of DEAE Fast Flow (Pharmacia LKB) equilibrated with buffer. FOR eluted from the column at
150 to 205 mM NaCl with a gradient (10 liters) from 0 to 0.5 M NaCl in
buffer. The flow rate was 20 ml/min, and 125-ml fractions were
collected. Fractions from this column with FOR activity were combined
(1.2 liters) and loaded directly onto a column (5 by 25 cm) of
hydroxyapatite (American International Chemical). The absorbed proteins
were eluted with a gradient (4.0 liters) from 0 to 0.2 M potassium
phosphate in buffer at a flow rate of 4.0 ml/min. Fractions of 100 ml
were collected, and FOR activity eluted as 55 to 140 mM phosphate was
applied. Fractions containing FOR activity were combined and
concentrated to approximately 12 ml by ultrafiltration with an Amicon
type PM-30 membrane. The concentrated sample of FOR was applied to a
column (6 by 60 cm) of Superdex 200 (Pharmacia LKB) equilibrated with
buffer containing KCl (200 mM). Fractions containing pure FOR as judged
by sodium dodecyl sulfate (SDS) gel electrophoresis were combined (150 ml), concentrated by ultrafiltration to approximately 5 ml, and stored as pellets under liquid N2.
Enzyme assays.
FOR activity was routinely determined at
80°C with formaldehyde (50 mM) as the substrate and benzyl viologen
(3 mM) as the electron acceptor (37). Where indicated in
Results, methyl viologen (3 mM) replaced benzyl viologen. The reduction
of P. furiosus ferredoxin by FOR was measured as
described previously (8). Crotonaldehyde (250 µM) and
glyceraldehyde-3-phosphate (300 µM) were used as substrates in the
assays for AOR and GAPOR, respectively. Results are expressed as
units per milligram of protein where 1 U equals the oxidation of 1 µmol of substrate/min/mg of protein. Hydroxycarboxylate-viologen
oxidoreductase (HVOR) assays were carried out at 80°C in 100 mM EPPS
[N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)] (pH 8.4) with either 2-hydroxy acids as substrates and oxidized
benzyl viologen (3 mM) as the electron acceptor or 2-keto acids as
substrates and reduced benzyl viologen (1 mM) as the electron donor
(45). For the latter assay, benzyl viologen was reduced with
sodium dithionite. Amino acid oxidoreductase assays were carried out at
both 50 and 80°C in 100 mM EPPS (pH 8.4) with various amino acids as
substrates (0.1 to 5 mM) and benzyl viologen (3 mM) as the electron
acceptor. Pyruvate formate lyase activity was determined by a two-step
assay modified from that previously described (31). The
assay mixture contained 100 mM EPPS (pH 8.4), sodium pyruvate (2 or 50 mM), coenzyme A (55 or 100 µM), and FOR (see below). This mixture was
incubated anaerobically at 80°C for 10 min and then rapidly cooled to
4°C. Formate was then measured at 37°C with an NAD-dependent
formate dehydrogenase from Saccharomyces cerevisiae (F8649;
Sigma Chemical Co., St. Louis, Mo.). NAD (1 mM) and formate
dehydrogenase (50 µg) were added to the cooled reaction mixture, and
NADH production was measured by the increase in absorbance at 340 nm. A
standard curve was prepared with 50 to 300 µM formate. For the HVOR,
amino acid oxidoreductase, and pyruvate formate lyase assays, the final
reaction mixture volumes were 2 ml and all assays contained either pure FOR (50 µg) or a cell extract of P. furiosus (50 µl
of ~15 mg/ml).
Other methods.
Iron (33), acid-labile sulfide
(11), cysteine (2, 43), pterin (51),
and protein (6, 34) were measured as described previously.
Molecular weight estimations (32, 37), plasma emission
spectroscopy (37), and N-terminal sequence analysis (13) were all carried out as described in the references.
Ferredoxin (2), AOR (36), and GAPOR
(38) were purified from P. furiosus as
described previously. Amino acid sequences were analyzed with the
Genetics Computer Group software program and MacVector (International Biotechnologies, Inc., New Haven, Conn.).
Nucleotide sequence accession numbers.
The DNA
sequences of the genes encoding FOR and the two putative
tungstoenzymes, WOR4 and WOR5, are available from GenBank under
accession no. AF102769, AF101432, and AF101433, respectively. The
DNA sequences for AOR and GAPOR (40) can be found in the
EMBL data bank under the accession no. X79777 and U74298, respectively.
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RESULTS |
Purification of FOR.
The ability of FOR to catalyze the
formaldehyde-dependent reduction of benzyl viologen was used to
detect the enzyme during its purification from P. furiosus cells. Cell extracts contained 4.3 ± 1.8 U of
formaldehyde-oxidizing activity per mg, which is much higher than that
found in cell extracts of T. litoralis (0.6 ± 0.2 U/mg). However, AOR (but not GAPOR) of P. furiosus
also uses formaldehyde as a substrate (36), so the apparent
FOR activity observed in the extracts of P. furiosus is
the sum of the formaldehyde-oxidizing activities of both FOR and
AOR. The AOR and FOR activities were easily separated by the
first chromatography step with DEAE-Sepharose. The FOR activity eluted
at 150 mM NaCl, whereas the AOR activity eluted much later at 210 mM
NaCl. FOR judged homogeneous by gel electrophoresis was obtained by two
further chromatography steps (hydroxyapatite and gel filtration). It
should be noted that substantial losses in activity were observed
throughout the purification procedure. The highest recovery of activity
(~6%) was obtained when sodium dithionite (2 mM), DTT (2 mM),
and glycerol (10%, vol/vol) were included in all buffers (see Table
1). This decreased to 2% if dithionite was omitted; hence,
P. furiosus FOR undergoes substantial inactivation
during purification, even under strictly anaerobic conditions. Possible
reasons for this are discussed below.
Approximately 50 mg of pure FOR was obtained from 500 g (wet
weight) of P. furiosus cells (Table
1). This compares with yields of 120 and
35 mg for AOR and GAPOR, respectively, from the same cell mass.
With benzyl viologen as the electron acceptor, the specific
activity of pure FOR ranged from 35 to 45 U/mg. These values are in the
same range as those reported for AOR (80 U/mg with crotonaldehyde
as the substrate) and GAPOR (25 U/mg with glyceraldehyde-3-phosphate as the substrate) when it was assayed under
standard conditions at 80°C. With methyl viologen rather than benzyl
viologen as the acceptor, the activities of both FOR (24 U/mg) and AOR
(8 U/mg) decreased while that of GAPOR (28 U/mg) was
unaffected. Based on the total amount of activity in a cell extract and assuming an intracellular volume of 5 µl/mg
(42), the intracellular concentrations of FOR, AOR, and
GAPOR were estimated to be 46, 112, and 112 µM, respectively.
These calculations are based on the holoenzyme forms (tetramer, dimer,
and monomer, respectively [see below]) and take into account the
formaldehyde oxidation activity of AOR in determining the FOR
concentration.
Molecular properties.
The apparent molecular weight of
P. furiosus FOR as determined by gel filtration was
275,000 ± 20,000 (average ± standard deviation) (data not
shown). After the enzyme was treated with SDS (1%, wt/vol) at 100°C
for 10 min, the sample after SDS gel electrophoresis gave rise to two
bands (Fig. 1) which corresponded to
molecular weights of 265,000 ± 20,000 and 68,000 ± 4,000. A single protein band corresponding to a molecular weight of 68,000 ± 4,000 was observed if the sample was heated with SDS at
100°C for 30 min (Fig. 1). These results suggest that FOR is a
homotetrameric protein (Mr, ~272,000) with
subunits with Mrs of ~68,000. The latter
value is similar to the subunit molecular weights reported for AOR
(66,630 from the gene sequence [29]) and GAPOR
(63,000 by gel filtration and electrophoresis [38]),
although in contrast to FOR, AOR is a dimer (10) while
GAPOR appears to be a monomer (38). The high purity of
P. furiosus FOR and the presence of a single subunit
were confirmed by N-terminal amino acid sequencing. This gave rise to a
single sequence which showed similarity to the N-terminal sequences of
AOR and GAPOR from P. furiosus (Fig. 2). The N-terminal sequence (29 residues)
of FOR was used to search the genomic sequence database of
P. furiosus, which is nearing completion by multiplex
sequencing methods (12). The N terminus matched exactly the
translated 5' end of one open reading frame (ORF), now termed
for, which contained 619 codons corresponding to a protein
with a molecular weight of 68,724 (see below), which is in good
agreement with that (68,000) estimated by biochemical analyses.
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FIG. 1.
SDS gel electrophoresis analysis of purified
P. furiosus FOR. Samples of FOR (8.5 mg/ml) were
incubated with an equal volume of SDS (1%, wt/vol) at 100°C for 10 min (lane 3) or 30 min (lane 2) prior to electrophoresis on a 10%
(wt/vol) acrylamide gel. Lane 1 contained marker proteins with the
indicated molecular masses (in kilodaltons) for (from top to bottom)
myosin,  -galactosidase, phosphorylase b, bovine serum
albumin, ovalbumin, and carbonic anhydrase.
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FIG. 2.
N-terminal amino acid sequence analysis of P. furiosus FOR and related enzymes. Abbreviations: Pf FOR,
P. furiosus FOR (this work); ES AOR, ES-1 AOR
(21); Pf AOR, P. furiosus AOR
(36); Tl FOR, T. litoralis FOR (37);
Cf CAR, Clostridium formicoaceticum carboxylic acid
reductase (49); Pf GAP, P. furiosus
GAPOR (38); Ct CAR, Clostridium
thermoaceticum carboxylic acid reductase ( -subunit)
(47); HVOR, P. vulgaris HVOR
(48). Identical amino acids are in boldface type and
underlined.
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Colorimetric analyses indicated that P. furiosus FOR
contained 3.8 ± 0.3 mol of Fe, 2.9 ± 0.5 g-mol of
acid-labile sulfide, and six cysteines ± 1 cysteine per subunit.
The last value agrees with that (six Cys/subunit) determined from the
amino acid sequence (see below). An elemental analysis of FOR by
inductively coupled plasma emission spectroscopy revealed the presence
(in gram-atoms per subunit) of 3.8 ± 0.3 mol of Fe, 0.9 ± 0.1 mol of W, 1.5 ± 0.1 mol of Mg, 1.4 ± 0.1 mol of P, and
0.4 ± 0.1 mol of Ca. No other metals were present in
significant amounts (>0.1 g-atom/subunit). The presence of
a pterin cofactor in FOR was confirmed by extracting it in the presence
of iodine and measuring the fluorescence of the resulting derivative.
The spectrum (not shown) was virtually identical to that previously
reported for the cofactor extracted from P. furiosus
AOR (36), as well as to those obtained from various
molybdenum-containing enzymes (except nitrogenase) when they are
treated in a similar fashion. Such spectra originate from the so-called
form A derivative of the pterin cofactor (41).
Catalytic properties.
FOR was purified by its abilities to
oxidize formaldehyde and reduce the artificial electron carrier
benzyl viologen. Under standard assay conditions (80°C, pH
8.4), the specific activity of pure FOR was dependent, for
reasons as yet unknown, upon the enzyme concentration in the assay
mixture, with the specific activity increasing fourfold as the enzyme
concentration increased from 0.01 to 0.15 mg/ml. Hence, to enable
meaningful comparisons, and unless otherwise stated, FOR was used at a
protein concentration of 0.025 mg/ml in all assay mixtures. The
activity of the pure enzyme with benzyl viologen as the electron
acceptor increased more or less linearly with pH over the pH range 5.5 (1.5 U/mg) to 10.0 (55 U/mg) at 80°C (data not shown). Specific
activity increased over the temperature range from 60°C (13 U/mg) to
90°C (58 U/mg) at pH 8.4 (data not shown). FOR activity, albeit low, was measurable at 25°C (~1 U/mg, pH 8.4). The enzyme was quite thermostable, with a time required for a 50% loss of activity (t50%) at 80°C of about 8 h (with 5 mg
of enzyme per ml in 100 mM EPPS, pH 8.4, containing 2 mM sodium
dithionite and 2 mM DTT). For comparison, the values for AOR and
GAPOR when they were treated under the same conditions were 15 min
and 1 h, respectively. Note that activity values at 80°C (and
above) were determined for all three enzymes from initial rates of
benzyl viologen reduction, which were measured over a period of 30 s or less.
For FOR under standard assay conditions, a linear Lineweaver-Burk
plot was obtained when the formaldehyde concentration was varied from
0.5 to 100 mM. The apparent Km and
Vmax values were 25 mM and 62 U/mg,
respectively. The former value suggests that formaldehyde is unlikely
to be the physiological substrate. As shown in Table
2, FOR also oxidized various
C1 to C3 aldehydes when they were used at
either low (0.3 mM) or high (50 mM) concentrations. The exceptions were
glyoxal, which inhibited the enzyme at concentrations above 1 mM (data
not shown), and methyl glyoxal, which was not oxidized at a detectable
rate. However, the apparent Km value, calculated
from linear double-reciprocal plots, for both of the most active of the
C1 to C3 aldehydes, acetaldehyde and
propionaldehyde (Table 2), was 60 mM (Table
3), suggesting that these reactions also
are not of physiological relevance. As shown in Table 2, FOR exhibited
low activity with butyraldehyde (C4) or crotonaldehyde (C4) as a substrate but was not active with
isovaleraldehyde (C5), indicating that the catalytic site
of the enzyme is accessible only to short-chain (C1 to
C3) aliphatic aldehydes. This notion was supported by a
lack of detectable activity with the aromatic substrates benzaldehyde,
salicaldehyde, and 2-furfuraldehyde. On the other hand, FOR did oxidize
short-chain aldehydes with associated aromatic groups, such as
phenylacetaldehyde, phenylpropionaldehyde, and indole-3-acetaldehyde
(Tables 2 and 3). In fact, the kinetic constants determined for
indoleacetaldehyde and phenylpropionaldehyde were comparable to those
obtained with acetaldehyde (Table 3), although the relatively high
apparent Km values (
15 mM) again suggest that
the oxidation of aromatic acetaldehyde derivatives is not the in vivo
function of FOR.
A clue to other potential substrates for P. furiosus
FOR came from the fact that the N-terminal sequences of members of the AOR family of tungstoenzymes, which includes FOR (Fig. 2), were previously reported (30) to have similarity with that of the molybdoenzyme HVOR. HVOR, which has been purified from Proteus vulgaris (48) and Clostridium tyrobutyricum
(3), has a broad substrate specificity and catalyzes the
reversible oxidation of 2-hydroxycarboxylic acids with benzyl viologen
as the electron carrier. However, P. furiosus FOR did
not oxidize lactate, 2-hydroxybutyrate, 2-hydroxyvalerate,
2-hydroxycaproate, or 1-ethyl-2-hydroxycaproate and it did
not reduce pyruvate, 2-ketobutyrate, 2-ketovalerate, 1-ethyl-3-ketovalerate, 1-ethyl-4-ketovalerate, or
2-ketocaproate (with these substrates being used at a 5 or 50 mM final
concentration). In addition, FOR was unable to oxidize amino acids
(glycine, alanine, serine, threonine, aspartate, glutamate, and
sarcosine) or formate (to CO2) with benzyl viologen or NAD
as the electron acceptor and it did not exhibit pyruvate formate lyase
activity. Furthermore, none of these activities were detected in a cell
extract of P. furiosus.
Thus, of all the compounds listed above, P. furiosus
FOR oxidizes only short-chain (at most C4) unsubstituted
aldehydes, but even these are poor substrates, as shown by the high
apparent Km values. Our attempts to uncover
related substrates for which the enzyme had a much greater affinity
were limited by the availability of substituted compounds of this type.
Nevertheless, FOR did catalyze the oxidation of succinate
semialdehyde (C4) at a reasonable rate and the apparent
Km value was almost an order of magnitude lower than that of propionaldehyde (Table 3). Moreover, while
butyraldehyde (C4) was oxidized by FOR only at
extremely high concentrations (Table 2), the presence of a
terminal aldehyde group, as in glutaric dialdehyde (C5),
resulted in a substrate that was oxidized at a rate comparable
to that obtained with formaldehyde and acetaldehyde but with an
apparent Km value more than 25-fold lower (25 versus 0.8 mM) (Table 3). Increasing the chain length to C6
(adipic acid semialdehyde, methyl ester) resulted in an
activity similar to that obtained with succinic semialdehyde (catalytic
constant [kcat] = 3.0 × 104
s
1) but with a much higher Km
value (30 mM). From these analyses we conclude that the true
substrate for FOR is therefore most likely a
C5 di- or semialdehyde, although its precise nature is still unclear.
The activity of FOR was largely unaffected when it was assayed in the
presence of various mono- and diacids (formate, acetate, succinate,
citrate) when they were used at concentrations of 10 mM. The exception
was glutarate, which resulted in ~15% inhibition under standard
assay conditions. At a higher concentration of the acids (100 mM), the
enzyme was inhibited by 35, 32, 36, 75, and 70% in the presence of
formate, acetate, succinate, glutarate, and citrate, respectively.
These data again suggest that C5- or C6-type
aldehydes might be physiologically relevant. With either formaldehyde
(50 mM) or glutaric dialdehyde (30 mM) as the substrate for FOR,
linear double-reciprocal plots were obtained with P. furiosus ferredoxin in place of benzyl viologen as the electron acceptor (concentration range, 10 to 200 µM). The apparent
Km value for the ferredoxin was approximately
100 µM (at pH 8.0) with both substrates. Over the pH range 6.0 to
10.0, the highest rate of ferredoxin reduction (coupled to formaldehyde
oxidation) was observed at pH 6.0. The rate decreased with increasing
pH and at pH 10.0 was about 30% of that observed at pH 6.0 (data not
shown). Thus, although the in vivo aldehyde substrates for AOR and
GAPOR are known, that for FOR remains unknown but, like AOR
and GAPOR, FOR utilizes ferredoxin as its physiological
electron carrier.
FOR was routinely purified in the presence of glycerol (10%, vol/vol)
as this appeared to stabilize the enzyme. That is, when FOR was
purified in the absence of this reagent, the final recovery of FOR
activity decreased by about twofold, as did the specific activity of
the pure enzyme. However, although glycerol is a potential substrate
analog, it had little effect on FOR activity. For example, when
glycerol-free FOR was incubated (at 23°C in 50 mM Tris-HCl buffer, pH
8.0) for up to 6 h with up to 40% (vol/vol) glycerol, there was
no significant loss of activity (determined in the absence of
glycerol). The enzyme was inhibited when 60% (vol/vol) glycerol was
used, as about 60% of the activity was lost after 6 h. Under the
same conditions, P. furiosus AOR was much more
sensitive to inhibition by glycerol, with a loss of 25 and 80% of the
initial activity after a 4-h incubation with 20 and 40% (vol/vol)
glycerol, respectively. P. furiosus GAPOR was even
more sensitive to inhibition by glycerol, with
t50%s of 30 and 10 min in the presence of 40 and 60% glycerol, respectively. Hence, although all three enzymes are
routinely purified in the presence of glycerol (10%, vol/vol),
significantly higher concentrations are required for inhibition.
P. furiosus FOR as purified under standard conditions
was oxygen sensitive, with the t50% being about
12 h when FOR was exposed to air (no activity was lost under
anaerobic conditions). For comparison, a similar value was obtained
with P. furiosus GAPOR, whereas AOR from
P. furiosus was much more sensitive to oxygen, with the
t50% being about 30 min in air. P. furiosus FOR was not inhibited when it was incubated for up to
24 h with iodoacetate, arsenite, or cyanide (each at a
concentration of 5 mM) prior to being assayed under standard conditions
in the absence of these reagents. In contrast, both AOR and GAPOR
of P. furiosus were inhibited by all of these reagents
under the conditions used for FOR. Thus, when AOR was incubated with
either potassium cyanide, sodium arsenite, or sodium iodoacetate (each at 5 mM), the t50% was 13 h, 10 min, or 5 min, respectively. With GAPOR under the same conditions, the
t50% was 30 min, 12 h, or 20 min,
respectively. Thus, while GAPOR is very sensitive to inhibition by
cyanide and AOR is readily inhibited by arsenite and iodoacetate, FOR
is not affected by these reagents.
Properties of sulfide-activated FOR.
A troubling aspect
concerning the characterization of P. furiosus FOR was
the significant loss of activity during purification. This loss
occurred even when the procedure was carried out under the most
rigorous of anaerobic conditions with reducing agents (DTT and sodium
dithionite) in all buffers. A clue to the mechanism involved was the
finding that the enzyme can be activated by sulfide. As shown in Fig.
3, incubation of FOR with excess
sodium sulfide (20 mM) and sodium dithionite (20 mM) at 23°C (pH 8.0)
resulted after a 6-h period in a >5-fold increase in specific
activity. No activation was observed even after 8 h if either
reagent was omitted. The degree of activation varied from preparation
to preparation, but all samples analyzed showed at least a threefold
increase in formaldehyde oxidation activity after a 6-h period and
thereafter showed little if any further increase, even after 24 h.
The time course and degree of activation were similar when sodium
dithionite was replaced by titanium(III) citrate (20 mM), but DTT (20 mM) was ineffective as the reductant in the activation reaction. When the enzyme was incubated with sulfide for 6 h and the sulfide was
then removed (by gel filtration with 50 mM Tris-HCl [pH 8.0] containing 2 mM sodium dithionite, 2 mM DTT, and 10% [vol/vol] glycerol), the specific activity of the resulting sulfide-free form of
the activated enzyme decreased by ~20% but this specific activity
remained unchanged even after a further 24 h under anaerobic conditions (in the absence of sulfide). The sulfide-free, activated form of FOR was much more sensitive to inactivation by oxygen than the
purified form, with a decrease in the t50% from 12 to about 4 h. Similarly, in contrast to the purified form, sulfide-activated FOR was sensitive to inhibition by cyanide. For
example, while untreated FOR was unaffected by a 4-day
incubation with cyanide (5 mM), the activated form (10 mg/ml in 50 mM
Tris-HCl, pH 8.0) lost approximately 70% of its activity after a
16-h incubation under the same conditions.
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FIG. 3.
Activation of P. furiosus FOR by
sulfide. The enzyme (10 mg/ml in 50 mM Tris-HCl, pH 8.0) was incubated
at 23°C with either sodium sulfide (20 mM, open squares), sodium
dithionite (20 mM, filled squares), or both (filled circles). At the
indicated times, samples were removed and the residual activities were
determined with formaldehyde as the substrate under standard assay
conditions.
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Thus, treating FOR with sulfide results in an enzyme that is more
active in catalyzing formaldehyde oxidation and that is more sensitive
to inactivation by both cyanide and oxygen. It seems reasonable to
suggest that the larger-than-expected decrease in activity of FOR
during purification was due, in substantial part, to loss of sulfide.
While the mechanisms of the sulfide-dependent effects are under study,
of more relevance to the present work was the possibility that the
substrate specificity of the sulfide-activated form of FOR was
different from that of the purified or native form of the enzyme.
Kinetic analyses were therefore conducted with three different batches
of each of the purified and sulfide-activated forms, and the results
are shown in Tables 2 and 3 (values are averages of at least six
determinations). As indicated, although the range of substrates used by
the two forms of the enzyme was the same, there were significant
changes in the kinetic properties of the activated enzyme. Thus, while
some of the substrates, like formaldehyde, were oxidized at a higher
rate by the sulfide-activated form of the enzyme, some were oxidized at
the same rate (glyoxal) or at a lower rate (formamide, propionaldehyde,
and crotonaldehyde) and phenylacetaldehyde was seemingly not oxidized
at all by the sulfide-activated form. Similarly, as shown in Table 3,
while the catalytic efficiencies of the sulfide-activated enzyme, as determined by the kcat/Km
values, were generally higher than those exhibited with the prepared
enzyme, this result was due mainly to the lower
Km values. The activated enzyme had higher
kcat values with only four of the seven
substrates tested, and these ranged between 136 and 209% of the values
obtained with the same substrate and the enzyme as prepared (Table 3).
Hence, while the sulfide-activated enzyme does show a large increase
(approximately fivefold) in activity with formaldehyde as the substrate
under standard assay conditions, relative to the activity of the
purified enzyme, this is obviously not the situation with all of the
substrates that this enzyme can utilize. At this point it is not clear
why the degree of activation depends on the substrate that is utilized. Notably, similar kinetic values were determined for the two enzyme forms with the best substrate found so far, glutaric dialdehyde (Table
3).
Gene sequences of related enzymes.
With the availability of
the complete amino acid sequence of FOR, the genomic sequence database
of P. furiosus (12), while still incomplete,
was also searched for related enzymes. The N-terminal sequence (45 residues) of P. furiosus GAPOR (38)
matched exactly the translated 5' end of one ORF, now termed
gor. The gene contained 653 codons corresponding to a
protein with a molecular weight of 73,850. This value is in reasonable
agreement with that (63,000) estimated by biochemical analyses
(38). The gene encoding AOR (aor) was located in
the database with the previously published sequence (29),
and the two nucleotide sequences were identical. The genes encoding FOR
(for), aor, and gor were spatially
separated on the genome, and except for the ORFs adjacent to the
previously identified cofactor-modifying (cmo) gene
(29), none of the ORFs immediately adjacent to the
for, aor, and gor genes appeared to have a role in the synthesis or function of these three tungstoenzymes in P. furiosus. The complete sequences of the three
enzymes are aligned in Fig. 4. FOR and
AOR are highly similar (61% similarity, 40% identity), with GAPOR
being somewhat less closely related (50% similarity and 23% identity
with FOR and 45% similarity and 25% identity with AOR).
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|
FIG. 4.
Alignment of the sequences of FOR, AOR, and GAPOR
and the putative gene products WOR4 and WOR5 from P. furiosus. Identical residues are boxed, and similar residues are
shaded.
|
|
A search of the genome database with the complete amino acid sequences
of FOR, AOR, and GAPOR revealed the presence of two additional
ORFs, and these appear to encode the fourth and fifth members of this
enzyme family. Tentatively termed wor4 and wor5 (to indicate genes encoding putative oxidoreductases within the tungstoenzyme family), these genes are also spatially separated from
those encoding the other three tungstoenzymes, and adjacent genes
appear to be unrelated (although obviously the functions of these
putative enzymes are as yet unknown). The wor4 and
wor5 genes encode 622 and 582 codons, corresponding to
proteins with molecular weights of 69,363 and 64,889, respectively, and
their sequences are also aligned in Fig. 4. The similarities
(identities shown in parentheses) of the sequence of the WOR4 protein
to those of FOR, AOR, and GAPOR are 57% (36%), 58% (37%), and
49% (25%), respectively, and those of the WOR5 protein are 56%
(33%), 58% (36%), and 49% (25%), respectively. Hence, both WOR4
and WOR5 appear to be more closely related to AOR and FOR than they are to GAPOR.
The complete genomes of several hyperthermophilic archaea are
now, or soon will be, available, and these were searched for relatives of FOR and other members of the tungstoenzyme family found in
P. furiosus. These genomes include those of
Pyrococcus horikoshii (19, 25), Pyrobaculum
aerophilum (15, 16), Methanococcus
jannaschii (9), and Archaeoglobus fulgidus
(28), all of which contained genes encoding putative
tungstoenzymes. The phylogenetic relationship between the amino acid
sequences of the five tungstoenzymes proposed for P. furiosus and their homologs in the other genome sequences of
various archaea is depicted in Fig. 5.
From the dendogram three distinct groups are apparent, and these
correspond to the three types of enzymes that have been purified and
characterized so far. Group I, or the FOR group, has homologous genes
in P. horikoshii and Pyrobaculum aerophilum as well as in P. furiosus and T. litoralis
(previously characterized). Group II enzymes have representatives in
P. horikoshii, A. fulgidus, and
Pyrobaculum aerophilum as well as in P. furiosus, while the group III enzyme, GAPOR, although it has
so far been purified only from P. furiosus, has close
homologs in P. horikoshii and M. jannaschii.
It should be noted that these sequences have been placed in their
respective groups based solely on similarity at the amino acid level,
but the corresponding enzymes are predicted to have the same function
as those of the previously characterized enzymes. As indicated in Fig.
5, there are also several sequences that do not fall into any of the
three groups, including WOR4 and WOR5 of P. furiosus,
and any existing relationship among them will have to await elucidation
of function through biochemical or genetic analyses.
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|
FIG. 5.
Phylogenetic relation of the amino acid sequences of
tungstoenzymes from P. furiosus and their homologs
found in genome databases. Sequences demarcated by brackets and Roman
numerals indicate groups. Asterisks denote amino acid sequence
homologous to a tungstoenzyme of known function, though the relevant
enzyme activity has not yet been determined in these organisms. Length
of the line represents distance in arbitrary units. Abbreviations: Ph,
P. horikoshii; Pf, P. furiosus; Mj,
M. jannaschii; Pa, Pyrobaculum aerophilum; Af,
A. fulgidus. The GenBank accession numbers of amino
acid sequences used in this figure are as follows: Ph GAPOR*,
g3130733; Mj GAPOR, U67559; Ph WOR5, g3131172; Ph WOR6, g3131173;
Ph WOR4, g3130250; Ph FOR*, g3257694; Ph AOR*, g3131315; Af AOR*,
g2648240; Af WOR3, g2650295; Af WOR4, g2650628; and Af WOR2, g2650571.
See the text for accession numbers of the P. furiosus
sequences. For Pa FOR*, Pa AOR*, and Pa WOR3 accession numbers,
see reference 16.
|
|
| |
DISCUSSION |
FOR is the third tungsten-containing, aldehyde-oxidizing enzyme,
in addition to AOR and GAPOR, to be characterized from
P. furiosus. Their cellular concentrations appear to be
comparable, at least in cells grown with maltose as the primary carbon
source. The Fe, W, P, and Mg content of FOR is similar to that of AOR (10, 36) and also GAPOR, but FOR also contains Ca
(apparently one atom/subunit) while GAPOR also contains Zn (two
atoms/subunit) (38). The function of Ca in FOR (and of Zn in
GAPOR) is not known. From crystallographic analysis, AOR is known
to contain one [4Fe-4S] cluster and a mononuclear tungstobispterin
cofactor (10). As shown in Fig.
6, the two pterin-binding motifs and all
four of the Cys residues in AOR are conserved in FOR and also in
GAPOR, indicating that they too contain one [4Fe-4S]
cluster and a mononuclear tungstobispterin cofactor. AOR also
contains two EXXH motifs, which coordinate a mononuclear metal
site, most likely iron, that bridges the two subunits. FOR lacks these
motifs (as does GAPOR, which is monomeric) and presumably lacks
subunit-bridging metal ions, a conclusion supported by its metal
content (Fig. 6). The sequence of the FOR of T. litoralis is
also available (29), and in spite of the low similarity in
the overall DNA sequences of P. furiosus and T. litoralis, it is virtually identical to the P. furiosus enzyme, with 92% similarity (87% identity) to its amino
acid sequence. The two putative tungstoenzymes in P. furiosus, WOR4 and WOR5, have molecular
weights comparable to those of FOR, AOR, and GAPOR, and in
its sequence, each contains a motif, separated by the appropriate
number of residues, that binds a single [4Fe-4S] cluster and a
bispterin site (Fig. 6). These enzymes also contain EXXH motifs but
they are not in the same location as those of AOR, suggesting that WOR4
and WOR5 lack the subunit-bridging metal ion found in AOR. In any
event, these two putative tungstoenzymes are clearly closely related
both in cofactor content and in structure to the three tungstoenzymes that have been purified from P. furiosus, although
their functions are obviously unknown.
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|
FIG. 6.
Alignments of the cofactor-binding motifs of FOR, AOR,
and GAPOR and of the putative gene products WOR4 and WOR5 from
P. furiosus. The numbers in parentheses indicate the
numbers of residues between the indicated motifs. See the text for
details.
|
|
Homologs of the three characterized tungstoenzymes and of the two
putative tungstoenzymes of P. furiosus were identified
in the genomes of other hyperthermophilic archaea. P. horikoshii has representative sequences of tungstoenzymes in
all three main groups, namely, FOR, AOR, and GAPOR, as well
as homologs of the putative P. furiosus WOR4 and WOR5
enzymes. This finding is consistent with the physiological similarities
between the two organisms (8, 19). In fact, P. horikoshii has a sixth member of the tungstoenzyme family, close
homologs of which two (WOR5 and WOR6) have not yet been identified in
the still incomplete genome of P. furiosus. It remains
to be seen if P. furiosus contains more than five
tungstoenzymes. Similarly, the presence of homologs of FOR and AOR,
both of which have proposed roles in peptide metabolism, in
Pyrobaculum aerophilum and A. fulgidus is
consistent with the reported ability of both organisms to use peptides
as a carbon source, even though their modes of energy conservation are
quite different (by respiring nitrate or oxygen and by sulfate
reduction, respectively). On the other hand, the finding of a
GAPOR-type sequence in M. jannaschii is unexpected. This
methanogenic organism is thought to grow only on
H2-CO2, and yet GAPOR is a key enzyme in
the glycolytic pathway of heterotrophic hyperthermophiles. Either
GAPOR has another function in methanogens or else M. jannaschii can grow on other carbon substrates, perhaps derived
from storage compounds.
The true role of FOR in P. furiosus also remains a
mystery, in spite of the extensive analyses of potential physiological substrates reported herein. Compared to formaldehyde, FOR (in both the
purified and sulfide-activated states [see below]) exhibited a higher
kcat/Km value only with
glutaric dialdehyde, a C5 compound, but this compound does
not lie on a known biochemical pathway. Various C4 to
C6 semialdehydes are involved in the metabolism of certain
amino acids such as Arg, Lys, and Pro (4, 20, 52), and
although such compounds are not commercially available, whether
relevant enzymes are present in cell extracts of P. furiosus is under study. Indeed, it should be noted that
P. furiosus appears to utilize AOR and GAPOR for
very different purposes, namely, in peptide catabolism and
glycolysis, respectively, yet these two enzymes have very
similar molecular properties and presumably are also closely related
phylogenetically. Thus, it is hard to rationalize a potential
physiological role for FOR based merely on its similarity to AOR
and GAPOR. Similarly, while an aldehyde-oxidizing enzyme with a
substrate specificity analogous to that of AOR has been found in
mesophilic clostridia (47, 49), an enzyme with the substrate
range of FOR has not been reported for any organism. A similar
situation will no doubt exist for the putative tungstoenzymes WOR4 and
WOR5, and elucidating their functions, particularly in the absence of
purified enzymes, will likely require a genetic approach.
Last, we turn to the finding that FOR is activated by incubation with
sulfide under reducing conditions. Although loss of sulfide can account
for the significant loss of formaldehyde oxidation activity observed
during the purification of FOR, it raises several issues, such as what
is the nature of the sulfide that is lost, why is the extent of sulfide
activation substrate dependent, and is sulfide activation a
physiologically relevant reaction? While there is no experimental
evidence to address the last question, the sulfide that is lost is
presumably associated with the catalytic site, and that site in the
activated and prepared forms of the enzyme presumably has different
reactivities with different substrates. By analogy with AOR
(10), FOR is expected to contain a tungstobispterin site
where the W atom is coordinated in part by four S atoms from the
dithiolene groups of two pterin molecules, with water, hydroxide, and/or a terminal oxo group completing the coordination sphere. Interestingly, mononuclear molybdopterin-containing enzymes such as xanthine oxidase lose sulfur as thiocyanate when the oxidized forms
are treated with cyanide (see, for example, reference
23). The resulting "desulfo" enzymes are
activated by treatment with sulfide, which is accomplished by
conversion of a terminal Mo==O species to Mo==S, which does not
involve the dithiolene sulfurs. So, is FOR as purified equivalent to
the desulfo state, and does sulfide activation involve W==O-to- W==S
conversion? It is noteworthy that upon sulfide activation, FOR became
more sensitive to inactivation by O2 and by cyanide,
suggesting that both may effect a W==S-to-W==O conversion. However,
for AOR, there was no evidence for a W=S bond either from
crystallography (10) or X-ray absorption spectroscopy (18). Thus, the precise mechanism by which sulfide activates FOR remains to be determined, and spectroscopic analyses using both
X-ray absorption and resonance Raman (see reference
17) of the enzyme are in progress to address these issues.
This research was supported by grants from the Department
of Energy (FG05-95ER20175) and the National Science Foundation
(BCS-9632657).
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Abstract
Introduction
Present address: Monsanto, St. Louis, MO 63198.
