Previous Article | Next Article 
Journal of Bacteriology, January 2001, p. 791-794, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.791-794.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Thermotoga maritima
Phosphofructokinases: Expression and Characterization of Two
Unique Enzymes
Yan-Huai R.
Ding,
Ron S.
Ronimus, and
Hugh W.
Morgan*
Thermophile Research Unit, Department of
Biological Sciences, The University of Waikato, Hamilton, New
Zealand
Received 5 June 2000/Accepted 25 October 2000
 |
ABSTRACT |
A pyrophosphate-dependent phosphofructokinase (PPi-PFK)
and an ATP-dependent phosphofructokinase (ATP-PFK) from
Thermotoga maritima have been cloned and characterized.
The PPi-PFK is unique in that the
Km and Vmax
values indicate that polyphosphate is the preferred substrate over
pyrophosphate; the enzyme in reality is a polyphosphate-dependent PFK.
The ATP-PFK was not significantly affected by common allosteric
effectors (e.g., phosphoenolpyruvate) but was strongly inhibited by
PPi and polyphosphate. The results suggest that the control
of the Embden-Meyerhof pathway in this organism is likely to be
modulated by pyrophosphate and/or polyphosphate.
| |
TEXT |
The Embden-Meyerhof (EM), or
glycolytic, pathway is nearly ubiquitous in all life forms, and enzymes
of the reaction sequence are highly conserved. One of the key and
definitive enzymes of the pathway is phosphofructokinase (PFK). In the
majority of organisms, ATP is the phosphoryl donor for the enzyme and
the reaction is a nonreversible step in the pathway. Due to its
position, PFK is usually allosterically regulated by intracellular
metabolites, e.g., phosphoenolpyruvate (PEP), GDP, and/or ADP
(27). PFK subtypes utilizing pyrophosphate
(PPi) as the phosphoryl donor, where the reaction
becomes more reversible and the enzyme is generally not subject to
allosteric control mechanisms, have also been described (16, 18,
25).
Thermotoga maritima is a non-spore-forming, rod-shaped
hyperthermophilic bacterium with an optimum growth temperature of
80°C and is phylogenetically classified in the order
Thermotogales. The phylogeny of the small-subunit rRNA shows
that this organism represents one of the deepest and most slowly
evolving lineages of bacteria (12). T. maritima
ferments various carbohydrates, including monosaccharides and
polysaccharides, primarily via the EM pathway, and ATP-dependent PFK
(ATP-PFK) activity in cell extracts has been reported (23,
24). The genome sequence of this organism indicated the presence
of another PFK gene, and sequence comparison showed homology to
PPi-dependent PFK (PPi-PFK)
enzymes (17). If both genes code for functional enzymes,
then Thermotoga would represent the unusual situation of an
organism possessing two distinct PFK activities. Because of its
phylogenetic position, the occurrence and origin of these genes are of
importance with respect to the origins of the EM pathway. This paper
describes the cloning, expression, and characterization of these
enzymes, both of which exhibit unusual features.
T. maritima strain 3109 was obtained from the Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH and grown in the medium described by Huber et al. (12). Escherichia
coli DH5
and expression plasmid pPROEX HTb were
obtained from Life Technologies. E. coli was grown at 30°C
with vigorous aeration (200 rpm) in Luria-Bertani broth supplemented
with ampicillin (100 µg ml
1) when
appropriate. The PFK assay was conducted essentially as described by
Ding et al. (6). Preparation of genomic DNA and the
alkaline lysis and cesium chloride gradient methods for large scale
plasmid DNA purification followed standard procedures
(21).
Construction of the PPi-PFK and ATP-PFK expression
clones.
The open reading frames representing the full-length
sequences of the PPi- and ATP-PFK genes were
amplified directly from genomic DNA from T. maritima. Primer
design was based on the nucleotide sequences of the 5'and 3' ends of
the putative PFK genes (17). For the
PPi-PFK gene, the forward primer, corresponding
to the N terminus, contained an upstream SfoI site (in bold)
and 5'-end spacer (5'-GGAA GGC GCC ATG GCT GAA AGA TTG
GGG ATA CTC G-3'), and the reverse primer, corresponding to the C
terminus, contained a flanking HindIII site (in bold)
and a 5'-end spacer (5'-GCTA AAG CTT TAT GGA AGC TCT
GTC GTA TGC CAG-3'). The primers for the ATP-PFK gene also
contained SfoI and HindIII sites, and their
sequences were 5'-GGCT GGC GCC ATG AAG AAG ATA GCA GTA
TAC-3' and 5'-CCA TAA GCT TTA TGA AAG CAT ATG TGC TAT TTC-3' for forward and reverse primers, respectively.
AmpliTaq Gold DNA polymerase was used for PCR (Perkin
Elmer). Both PCR products for the two genes (pfp and
pfk) were of the sizes predicted from their nucleotide
sequences, approximately 1,200 and 950 bp, respectively
(17). These products were sequenced to confirm their
identity (1) and then cloned into the expression vector after restriction digestion with SfoI and
HindIII, followed by ligation with T4 DNA ligase using
standard protocols (21). The ligation mixture containing
restriction enzyme-digested plasmid and PCR product was used to
transform E. coli strain DH5 by electroporation, according to the manufacturer's instructions (Gene Pulser; Bio-Rad). Screening of the clones for those with inserts was carried out through
alkaline lysis miniprep plasmid isolation (21) followed by
restriction enzyme analysis.
Expression, purification, and characterization of the recombinant
PPi- and ATP-PFKs.
Flask cultures of the recombinant
E. coli clones were grown at 30°C in 700 ml of
Luria-Bertani broth plus 100 µg of ampicillin ml1 and were induced with 1 mM
isopropyl-
-D-thiogalactoside when the culture
optical density at 600 nm reached approximately 0.6. After 5 h of
induction, the cells were harvested by centrifugation and sonicated,
and the cell lysate was incubated for 40 min at 80°C. Further
purification of the enzymes from the supernatant was performed using a
3.0-ml column of nickel nitrilotriacetic acid resin and elution
following the manufacturer's instructions (Life Technologies). Single
bands were obtained for each of the nickel nitrilotriacetic acid
resin-purified proteins on denaturing sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, indicating
a high degree of purity. The estimated molecular weights for the
PPi- and ATP-PFK proteins from SDS-PAGE were
approximately 48,000 and 38,000, respectively (Table
1), which is in close agreement with the
molecular masses derived from the amino acid sequences (including the
N-terminal histidine tag, which is approximately 2 kDa). The conceptual
translation masses of the full-length open reading frames of the
pfp and pfk genes are 46,403 Da and 34,447 Da for
the PPi- and ATP-PFKs, respectively. The
recombinant PPi-PFK had a molecular mass of 96 kDa as determined by its elution during gel filtration chromatography,
which suggests that the active molecule exists as a homodimer. In
contrast, the molecular mass of the ATP-PFK was 200 kDa, and thus a
homotetramer is the most probable quaternary structure.
Both recombinant proteins showed enzyme activity specific for their
expected phosphoryl donors; thus, the PP
i-PFK was
active
with PP
i but had no activity with ATP, and
vice versa for the
ATP-PFK.
Thermotoga is thus confirmed as
the only prokaryote reported
which possesses two different functional
phosphoryl donor subtypes
of PFK. As expected, the enzymes were
extremely thermostable,
with half-lives for both being greater than
5 h at 90°C in phosphate
buffer.
PFKs generally have a requirement for a low concentration (<100 mM) of
either sodium or potassium ions for optimal activity
(
5,
20,
27). For the
Thermotoga enzymes, the requirement
for
potassium ions and the stimulation of activity by their presence
was
more pronounced. The optimum concentration of K
+
for both enzymes was 175 mM, and activity for the ATP- and
PP
i-PFKs
fell 50 and 60%, respectively, when KCl
was omitted from the reaction
mixture. It is possible that this
requirement reflects the marine
environment from which the organism was
isolated. Magnesium ions
were required for optimal activities of both
enzymes with Co
2+, Mn
2+,
and Ni
2+ being able to substitute for
Mg
2+ with the PP
i-PFK and
Mn
2+ and Fe
2+ being able to
substitute for Mg
2+ with the ATP-PFK (Table
1).
Both
Thermotoga enzymes were extremely
sensitive to
Cu
2+ and Zn
2+ (Table
1).
This sensitivity was also found within the
Dictyoglomus thermophilum native and recombinant PP
i-PFKs
(
6,
7) and
the archaeal
Desulfurococcus
amylolyticus ATP-PFK (
5).
The
Thermotoga PP
i-PFK was unique in
that it exhibited higher activity with tripolyphosphate
(PPP
i) and polyphosphate (poly-P)
as phosphoryl
donors than with PP
i as the donor, and the
apparent
Km and
Vmax values (Table
2)
indicate
that the
Thermotoga PP
i-PFK
functions
as a poly-P-dependent PFK. This is the first report
of a PFK with such
characteristics. The PP
i-PFK catalyzes a
typically
reversible reaction, but with the
Thermotoga
enzyme the pH optima
for the forward and reverse reactions are
unusually close; pH
5.6 to 5.8 for the forward reaction and pH 5.6 to
6.8 for the
reverse reaction (Table
1). In general, other
PP
i-PFKs have a
pH difference of up to one unit
between the forward and reverse
reactions. Similar to other
PP
i-PFKs, the
Thermotoga
PP
i-PFK exhibited
essentially no response to
traditional allosteric effectors, and
presumably the reaction direction
and rate (due only to the PP
i-PFK)
are dictated
simply by the concentrations of intracellular metabolites
and the level
of the enzyme.
The
Thermotoga ATP-PFK displayed the highest activity with
ATP as the phosphoryl donor (Table
3) but had significant activity
when
this was replaced by GTP, UTP, CTP, and TTP. No activity
was detected
with either PP
i, PPP
i,
poly-P, or ADP as the phosphoryl
donor (Table
1). The pH optimum for
the ATP-PFK was between 7.2
and 8.0 (which is more likely to reflect
the intracellular pH
of the organism). The ATP-PFK showed no
significant response to
the common allosteric regulators. Thus,
activity was only slightly
inhibited by citrate at 1.0 mM, and PEP
concentrations up to 5
mM did not affect the normal hyperbolic kinetic
curve for fructose
6-phosphate (F-6-P). The allosteric response
of the ATP-PFKs from
E. coli and
Bacillus
stearothermophilus is potentially controlled
by a glutamic acid
residue at position 187 (E
187) via the
binding of PEP (
2,
8,
22). Sequence alignment
shows that
the
Thermotoga ATP-PFK also possesses an equivalent
E
187 residue, but the biochemical properties from
this characterization
suggest that PEP is not vital for regulating the
Thermotoga enzyme
and thus probably does not regulate
glycolysis in this organism
(
7). ADP had opposing effects
on ATP-PFK activity, as the enzyme
was slightly activated at a low
concentration of ADP (129% at
0.05 mM) and partially inhibited at
higher concentrations (70%
at 1.0 mM), but the magnitude of these
effects does not reflect
allosteric
control.
Surprisingly, the
Thermotoga ATP-PFK activity was strongly
inhibited by PP
i, PPP
i, or
poly-P (
n = 15 ± 3) at concentrations
of less
than 0.10 mM and under conditions in which chelation effects
on
available Mg
2+ could be excluded (Table
4; Fig.
1). In particular, activity
was strongly
inhibited by both PP
i and poly-P at
concentrations
reported to be common in bacteria (10 to 100 µM)
(
14). Interestingly,
the inhibition of ATP-PFK activity by
PP
i could be partially alleviated
by the presence
of nucleotide diphosphates, i.e., ADP, GDP, or
TDP (Table
5). This type of allosteric control has
not previously
been reported, and it seems that
PP
i and/or poly-P might replace,
either partially
or fully, the function of PEP and other potential
modulators within
this organism. Although nonallosteric ATP-PFKs
have been identified in
other organisms, including
D. amylolyticus (
5,
9),
Trypanosoma brucei (
15), and
Lactobacillus bulgaricus (
4), the responses of
these enzymes to PP
i and poly-P have
not been
investigated.
Role of poly-P.
Both Thermotoga enzymes have unique
properties related to poly-P: it is a preferred substrate for the
PPi-PFK and an allosteric regulator for the
ATP-PFK. Poly-P is a component of volcanic condensates and deep-oceanic hydrothermal vents, and it is ubiquitously distributed in all living organisms (13) and possibly played a role in
the prebiotic evolution of metabolism (3, 29).
Significantly, poly-P has been used as an alternate phosphoryl and/or
energy source to ATP for other enzymes involved with glucose
metabolism. For example, poly-P-dependent glucokinase activity has been
observed in Mycobacterium tuberculosis (11) and
Propionibacterium freudenreichii (26,
28), and a poly-P-fructokinase has been found in
Mycobacterium phlei (28). The
poly-P-glucokinase from P. freudenreichii was particularly responsive to phosphoester chain length, with the apparent
Km declining from 4.3 µM to 0.2 nM for
polymer lengths of 30 and 724 residues, respectively (28).
The PPi-PFK from Thermotoga
demonstrated a similar, though less pronounced, effect, with a decline
in Km values from 67 to 3.8 µM as
phosphoester chain length increased from 2 to 18. In contrast, the
PPi-PFKs from D. thermophilum and
Spirochaeta thermophila favor the pyrophosphate substrate
(7, 20).
The results presented here indicate that the control of the EM pathway
in
Thermotoga may be mediated by a quite different
mechanism
than that conventionally found, where the activity of
ATP-PFK is
allosterically controlled by either PEP, ADP, AMP,
F-2,6-P
2, citrate, succinate, or a combination of
these. For glycolysis
to proceed utilizing the ATP-PFK, the
PP
i and poly-P concentrations
would have to
remain low (<100 µM). If poly-P accumulated and/or
the pH fell, then
the ATP-PFK would be inhibited and the PP
i-PFK
activity would predominate. Poly-P is regarded as ubiquitous in
all
tested organisms (
13,
14) and is present at concentrations
above that needed to inhibit the ATP-PFK. Interestingly, no gene
encoding a poly-P kinase has been identified in the genome of
Thermotoga, though in other organisms other enzymes have
also
been implicated in the synthesis of poly-P, e.g., adenylate kinase
in
Acinetobacter johnsonii (
19) and an acetate
kinase in
E. coli (
10). Possibly, the
PP
i-PFK could produce poly-P by means
of the
reverse reaction at intracellular pH values between 6.0
and 7.0. The
presence of ATP-PFK activity in cell extracts of
Thermotoga
has been reported (
23,
24). We found both
PP
i-PFK
and ATP-PFK activities in cell extracts
if the assay pH was adjusted
to the optimum for each enzyme (results
not shown), so the enzymes
appear to be expressed simultaneously. The
intracellular concentration
of PP
i and poly-P and
the internal pH of
Thermotoga are unknown,
but it will be
important to determine these if the control of
glycolysis in
Thermotoga is to be understood. In summary,
Thermotoga appears to be unique in that it contains the
genes for two distinct
PFKs and both genes can express functional
enzymes. Both enzymes
have unique properties, in particular, their
responses to PP
i and poly-P, and it is likely
that these metabolites may play a
central role in the control of
glucose metabolism in this
organism.
| |
ACKNOWLEDGMENTS |
We thank the Royal Society Marsden Science Foundation and the
University of Waikato for its financial support during the course of
this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Thermophile
Research Unit, The University of Waikato, Private Bag 3105, Hamilton, New Zealand. Phone: 64-7-8388266. Fax:
64-7-8384324. E-mail: h.morgan{at}waikato.ac.nz.
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
W. Gish,
W. Miller,
W. Myers, and D. L. Lipman.
1997.
Basic local alignment search tool.
J. Mol. Biol.
215:403-410.
|
| 2.
|
Auzat, I.,
G. Le Bras,
P. Branny,
F. D. L. Torre,
B. Theunissen, and J.-R. Garel.
1994.
The role of Glu 187 in the regulation of phosphofructokinase by phosphoenolpyruvate.
J. Mol. Biol.
235:68-72[Medline].
|
| 3.
|
Baltscheffsky, H.
1996.
Energy conversion leading to the origin and early evolution of life: did inorganic pyrophosphate precede adenosine triphosphate?, p. 1-9.
In
H. Baltscheffsky (ed.), Origin and evolution of biological energy conversion. VCH Publishers, New York, N.Y.
|
| 4.
|
Branny, P.,
F. De La Torre, and J.-R. Garel.
1993.
Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus.
J. Bacteriol.
175:5344-5349[Abstract/Free Full Text].
|
| 5.
|
Ding, Y.-H. R.
2000.
Phosphofructokinases from extremely thermophilic microorganisms. Ph.D. thesis.
The University of Waikato, Hamilton, New Zealand.
|
| 6.
|
Ding, Y.-H. R.,
R. S. Ronimus, and H. W. Morgan.
1999.
Purification and properties of the pyrophosphate-dependent phosphofructokinase from Dictyoglomus thermophilum Rt46 B.1.
Extremophiles
3:131-137[CrossRef][Medline].
|
| 7.
|
Ding, Y.-H. R.,
R. S. Ronimus, and H. W. Morgan.
2000.
Sequencing, cloning, and high-level expression of the pfp gene, encoding a PPi-dependent phosphofructokinase from the extremely thermophilic eubacterium Dictyoglomus thermophilum.
J. Bacteriol.
182:4661-4666[Abstract/Free Full Text].
|
| 8.
|
Evans, P. R., and P. J. Hudson.
1979.
Structure and control of phosphofructokinase from Bacillus stearothermophilus.
Nature
279:500-504[CrossRef][Medline].
|
| 9.
|
Hansen, T., and P. Schönheit.
2000.
Purification and properties of the first-identified, archaeal, ATP-dependent 6-phosphofructokinase, an extremely thermophilic non-allosteric enzyme, from the hyperthermophilic Desulfurococcus amylolyticus.
Arch. Microbiol.
173:103-109[CrossRef][Medline].
|
| 10.
|
Hardoyo,
K. Yamada,
A. Muramatsu,
Y. Anbe,
J. Kato, and H. Ohtake.
1994.
Molecular genetics of polyphosphate accumulation in Escherichia coli, p. 209-214.
In
A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in microorganisms: cellular and molecular biology. ASM Press, Washington, D.C.
|
| 11.
|
Hsieh, P.-C.,
B. C. Shenoy,
J. E. Jentoft, and N. F. B. Phillips.
1993.
Purification of polyphosphate and ATP glucose phosphotransferase from Mycobacterium tuberculosis H37Ra: evidence that poly(P) and ATP glucokinase activities are catalyzed by the same enzyme.
Protein Expr. Purif.
4:76-84[CrossRef][Medline].
|
| 12.
|
Huber, R.,
T. A. Langworthy,
H. Konig,
M. Thomm,
C. R. Woese,
U. B. Sleytr, and K. O. Stetter.
1986.
Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90oC.
Arch. Microbiol.
144:324-333[CrossRef].
|
| 13.
|
Kornberg, A.
1995.
Inorganic polyphosphate: toward making a forgotten polymer unforgettable.
J. Bacteriol.
177:491-496[Abstract/Free Full Text].
|
| 14.
|
Kulaev, I. S.
1979.
The biochemistry of inorganic polyphosphates.
John Wiley & Sons, Inc., New York, N.Y.
|
| 15.
|
Michels, P. A. M.,
N. Chevalier,
F. R. Opperdoes,
M. H. Rider, and D. J. Rigden.
1997.
The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei must have evolved from an ancestral pyrophosphate-dependent enzyme.
Eur. J. Biochem.
250:698-704[Medline].
|
| 16.
|
Morgan, H. W., and R. S. Ronimus.
1998.
Pyrophosphate-dependent phosphofructokinase in thermophilic and non-thermophilic microorganisms, p. 269-278.
In
M. W. Adams, and J. W. Wiegel (ed.), Thermophiles: the key to molecular evolution and the origin of life. Taylor and Francis, London, United Kingdom.
|
| 17.
|
Nelson, K. E.,
R. A. Clayton,
S. R. Gill,
M. L. Gwinn,
G. R. Dodson,
D. D. Haft,
K. Hickey,
J. D. Peterson,
W. C. Nelson,
K. A. Ketchum,
L. McDonald,
T. R. Utterback,
J. A. Malek,
K. D. Linher,
M. M. Garrett,
A. M. Steward,
M. D. Cotton,
M. S. Pratt,
C. A. Phillips,
D. Richardson,
J. Heidelberg,
G. G. Sutton,
R. D. Fleischmann,
J. A. Eisen,
O. White,
S. L. Salzberg,
H. O. Smith,
J. C. Venter, and C. M. Fraser.
1999.
Evidence for lateral gene transfer between archaea and bacteria from genome sequence of Thermotoga maritima.
Nature
399:323-329[CrossRef][Medline].
|
| 18.
|
Reeves, R. E.,
D. J. South,
H. J. Blytt, and L. G. Warren.
1974.
Pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase.
J. Biol. Chem.
249:7737-7741[Abstract/Free Full Text].
|
| 19.
|
Resnick, S. M., and A. J. Zehnder.
2000.
In vitro ATP regeneration from polyphosphate and AMP by polyphosphate:AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A.
Appl. Environ. Microbiol.
66:2045-2051[Abstract/Free Full Text].
|
| 20.
|
Ronimus, R. S.,
H. M. Morgan, and Y.-H. R. Ding.
1999.
Phosphofructokinase activities within the order Spirochaetales and the characterization of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila.
Arch. Microbiol.
172:401-406[CrossRef][Medline].
|
| 21.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 22.
|
Schirmer, T., and P. R. Evans.
1990.
Structural basis of the allosteric behaviour of phosphofructokinase.
Nature
343:140-145[CrossRef][Medline].
|
| 23.
|
Schröder, C.,
M. Selig, and P. Schönheit.
1994.
Glucose fermentation to acetate, CO2, and H2 in the anaerobic hyperthermophilic eubacterium Thermotoga maritima: involvement of the Embden-Meyerhof pathway.
Arch. Microbiol.
161:460-470[CrossRef].
|
| 24.
|
Selig, M.,
K. B. Xavier,
H. Santos, and P. Schönheit.
1997.
Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in the hyperthermophilic archaea and the bacterium Thermotoga.
Arch. Microbiol.
167:217-232[Medline].
|
| 25.
|
Siebers, B.,
H. P. Klenk, and R. Hensel.
1998.
PPi-dependent phosphofructokinase from Thermoproteus tenax, an archaeal descendant of an ancient line in phosphofructokinase evolution.
J. Bacteriol.
180:2137-2143[Abstract/Free Full Text].
|
| 26.
|
Uryson, S. O., and I. S. Kulaev.
1968.
The presence of polyphosphate glucokinase in bacteria.
Dokl. Akad. Nauk SSSR
183:957-959[Medline].
|
| 27.
|
Uyeda, K.
1979.
Phosphofructokinase.
Adv. Enzymol.
48:193-241.
|
| 28.
|
Wood, H. G., and J. E. Clark.
1988.
Biological aspects of inorganic polyphosphates.
Annu. Rev. Biochem.
57:235-260[CrossRef][Medline].
|
| 29.
|
Yamagata, Y.,
H. Watanabe,
M. Saitoh, and T. Namba.
1991.
Volcanic production of polyphosphates and its relevance to prebiotic evolution.
Nature
352:516-519[CrossRef][Medline].
|
Journal of Bacteriology, January 2001, p. 791-794, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.791-794.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Muller, M., Lee, J. A., Gordon, P., Gaasterland, T., Sensen, C. W.
(2001). Presence of Prokaryotic and Eukaryotic Species in All Subgroups of the PPi-Dependent Group II Phosphofructokinase Protein Family. J. Bacteriol.
183: 6714-6716
[Abstract]
[Full Text]
Top
Abstract
Text
