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Thermophiles 2003
Embden–Meyerhof–Parnas and Entner–Doudoroff
pathways in Thermoproteus tenax: metabolic
parallelism or specific adaptation?
H. Ahmed, B. Tjaden, R. Hensel and B. Siebers1
Department of Microbiology, University of Duisburg-Essen, Universitätsstr.5, 45117 Essen, Germany
Abstract
Genome data as well as biochemical studies have indicated that – as a peculiarity within hyperthermophilic
Archaea – Thermoproteus tenax uses three different pathways for glucose metabolism, a variant of the
reversible EMP (Embden–Meyerhof–Parnas) pathway and two different modifications of the ED (Entner–
Doudoroff) pathway, a non-phosphorylative and a semi-phosphorylative version. An overview of the three
different pathways is presented and the physiological function of the variants is discussed.
Introduction
The hyperthermophilic Crenarchaeote Thermoproteus tenax
is able to grow chemolithoautotrophically on CO2 , H2 and
sulphur, as well as chemo-organoheterotrophically in the
presence of sulphur and organic compounds such as glucose
and starch [1,2]. Early studies indicated that – as a peculiarity
within hyperthermophilic Archaea – T. tenax uses at least
two different pathways for glucose catabolism, a variant of
the EMP (Embden–Meyerhof–Parnas) pathway and the nonphosphorylative ED (Entner–Doudoroff) pathway [3–6].
In the course of the T. tenax genome-sequencing project
additional, unexpected gene homologues were identified giving new insights into various facets of archaeal carbohydrate
metabolism [7].
Variant of the EMP pathway in T. tenax
The variant of the EMP pathway is characterized by
(i) a hexokinase with reduced allosteric potential [8], (ii) a
non-allosteric, reversible PPi -dependent phosphofructokinase [9], (iii) three different GAP (glyceraldehyde 3phosphate)-converting enzymes, a classical, phosphorylating GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
[10,11], GAPN (a non-phosphorylating, highly allosteric
GAPDH) [12,13] and GAPOR (a ferredoxin-dependent
glyceraldehyde-3-phosphate oxidoreductase) [14], and
(iv) three enzymes for phosphoenolpyruvate and pyruvate
interconversion, a catabolic pyruvate kinase with low allosteric potential [15], an anabolic PEPS (phosphoenolpyruvate
synthetase) and a reversible PPDK (pyruvate phosphate
dikinase) [16,17]. Enzyme as well as transcript studies indicate that regulation of the EMP pathway takes place on
Key words: Archaea, central carbohydrate metabolism, hyperthermophile.
Abbreviations used: EMP, Embden–Meyerhof–Parnas; ED, Entner–Doudoroff; GAP, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAPN, non-phosphorylating, highly allosteric GAPDH; GAPOR, ferredoxin-dependent glyceraldehyde-3-phosphate
oxidoreductase; KDG, 2-keto-3-deoxy gluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate.
1
To whom correspondence should be addressed (e-mail [email protected]).
the protein and gene level [8,11,15,18] and suggest that –
in contrast to the classical pathway – the main control
point is shifted to the level of GAP. GAPN, like GAPOR,
catalyses the irreversible, non-phosphorylating oxidation of
GAP to 3-phosphoglycerate and thus both represent true
catabolic enzymes, which differ in cosubstrate specificity
and substitute for GAPDH and phosphoglycerate kinase.
The highly allosteric GAPN is activated in the presence
of effectors such as glucose 1-phosphate, AMP or ADP,
thus forcing the catabolic flux under conditions which are
characterized by the phosphorolytic degradation of storage
compounds (glycogen) and low energy charge of the cell. The
classical, phosphorylating GAPDH catalyses the conversion
of GAP to 1, 3-diphosphoglycerate, and transcript as well as
enzyme studies indicate that GAPDH and phosphoglycerate
kinase are of predominant importance for carbohydrate
anabolism.
Modifications of the ED pathway in T. tenax
13
C-labelling studies with growing cells [4] and cell
suspensions [6] demonstrated that in T. tenax an ED pathway
in addition to the variant of the EMP pathway is in operation.
Enzymic studies with cell-free extracts [3,5] and the
determination of characteristic intermediates identified
the non-phosphorylative version of the ED pathway, which
was described originally for Sulfolobus solfataricus [19] and
Thermoplasma acidophilum [20]. In this version of the
ED pathway, phosphorylation takes place only at the level
of glycerate and thus KDG (2-keto-3-deoxy gluconate)
and glyceraldehyde, generated by KDG aldolase, are the
characteristic intermediates of the pathway [3]. Surprisingly,
an unusual ED cluster was identified in the T. tenax genome
which comprises genes coding for gluconate dehydratase,
KD(P)G [2-keto-3-deoxy-(6-phospho-)gluconate] aldolase, KDG kinase and glucan-1,4-α-glucosidase. The identity
of the ED genes could be confirmed by biochemical
characterization of their encoded products after expression in
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Escherichia coli [7]. However, contrary to the S. solfataricus
KDG aldolase [21], the T. tenax enzyme is not only specific
for KDG or glyceraldehyde and pyruvate but also uses
KDPG or GAP and pyruvate as substrates and thus represents a KD(P)G aldolase. This finding and the presence of
the KDG kinase indicates that also the semi-phosphorylative version of the ED pathway, which has been assumed
to be characteristic for Haloarchaea, is active in hyperthermophiles. Thus, T. tenax obviously uses a variant of the
reversible EMP pathway and two different modifications
of the ED pathway (a non-phosphorylative and a semiphosphorylative version) for carbohydrate catabolism [7].
Metabolic parallelism or specific
adaptation
At the moment, we can only speculate about the physiological
meaning of the different pathways, since nothing is known
about the regulation of the ED pathways at the protein
and gene levels. However, the organization of the ED
genes coding for KDG kinase and KDG aldolase together
with a gene homologue for glucan-1,4-α-glucosidase in one
operon indicates a central role of the ED modifications in
the hydrolytic degradation of polysaccharides (e.g. glycogen
[22]). In contrast, the EMP pathway seems to be involved
in the phosphorolytic glycogen degradation by glycogen
phosphorylase in T. tenax, which has been characterized
recently [7]. Further, the energy demand seems to have a
strong influence on the selection of the different pathways.
Whereas the net ATP gain of the EMP variant is 1 (taking
into account that PPi – the phosphoryl donor of the
phosphofructokinase – is a waste product of the cell), no ATP
is generated by the two modifications of the ED pathway.
From these first hints we conclude that the different pathways do not represent a metabolic parallelism, but allow the
organism to respond to changing physiological needs of
the cell.
Additionally, the presence of different pathways may play
an important role for thermoadaptation. The half-lives of
intermediates (GAP, 14.5 min; dihydroxyacetone phosphate,
79.4 min; 1,3-diphosphoglycerate, 1.6 min; all at 60◦ C) suggest that the stability of intermediates represents the bottleneck for thermoadaptation [8]. Whereas the EMP and the
semi-phosphorylative ED pathways avoid the formation
of the extremely heat-labile 1,3-diphosphoglycerate by the
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one-step conversion of GAP to 3-phosphoglycerate via
GAPN or GAPOR, the non-phosphorylative ED variant
would additionally circumvent the formation of the two
other heat-labile intermediates GAP and dihydroxyacetone
phosphate. Therefore, the non-phosphorylative ED pathway
might be appropriate for growth at the upper temperature
range. From this first evidence we conclude that the
various pathways for carbohydrate metabolism do not reflect
metabolic parallelism but represent a measure for ‘metabolic
thermoadaptation’.
References
1 Fischer, F., Zillig, W., Stetter, K.O. and Schreiber G. (1983)
Nature (London) 301, 511–513
2 Zillig, W., Stetter, K.O., Schäfer, W., Janekovic, D., Wunderl, S., Holz, I.
and Palm, P. (1981) Zbl. Bakt. Hyg., I. Abt. Orig. C 2, 205–227
3 Siebers, B. and Hensel, R. (1993) FEMS Microbiol. Lett. 111, 1–8
4 Siebers, B., Wendisch, V.F. and Hensel, R. (1997) Arch. Microbiol. 168,
120–127
5 Selig, M. and Schönheit, P. (1994) Arch. Microbiol. 162, 286–294
6 Selig, M., Xavier, K.B., Santos, H. and Schönheit, P. (1997)
Arch. Microbiol. 167, 217–232
7 Siebers, B., Tjaden, B., Michalke, K., Gordon, P., Sensen, C.W., Zibat, A.,
Klenk, H.-P., Schuster, S.C. and Hensel, R. (2004) J. Bacteriol. 186,
in the press
8 Dörr, Ch., Zaparty, M., Tjaden, B., Brinkmann, H. and Siebers, B. (2003)
J. Biol. Chem. 278, 18744–18753
9 Siebers, B., Klenk, H.-P. and Hensel, R. (1998) J. Bacteriol. 180,
2137–2143
10 Hensel, R., Laumann, S., Lang, J., Heumann, H. and Lottspeich, F. (1987)
Eur. J. Biochem. 170, 325–333
11 Brunner, N.A., Siebers, B. and Hensel, R. (2001) Extremophiles 5,
101–109
12 Brunner, N.A., Brinkmann, H., Siebers, B. and Hensel, R. (1998)
Biochemistry 273, 6149–6156
13 Pohl, E., Brunner, N.A., Wilmanns, M. and Hensel, R. (2002) J. Biol. Chem.
277, 19938–19945
14 Van der Oost, J., Schut, G., Kengen, S.W.M., Hagen, W.R., Thomm, M. and
De Vos, W.M. (1998) J. Biol. Chem. 273, 28149–28154
15 Schramm, A., Siebers, B., Tjaden, B., Brinkmann, H. and Hensel, R.
(2000) J. Bacteriol. 182, 2001–2009
16 Siebers, B. (1995) Ph.D. Thesis, University of Essen-Duisburg, Essen
17 Tjaden, B. (2003) Ph.D. Thesis, University of Essen-Duisburg, Essen
18 Siebers, B., Brinkmann, H., Dörr, C., Tjaden, B., Lilie, H., Van der Oost, J.
and Verhees, C.H. (2001) J. Biol. Chem. 276, 28710–28718
19 De Rosa, M., Gambacorta, A., Nicolaus, B., Giardina, P., Poerio, E. and
Buonocore, V. (1984) Biochem. J. 224, 407–414
20 Budgen, N. and Danson, M.J. (1986) FEBS Lett. 196, 207–210
21 Buchanan, C.L., Connaris, H., Danson, M.J., Reeve, C.D. and Hough, D.W.
(1999) Biochem. J. 343, 563–570
22 König, H., Skorko, R., Zillig, W. and Reiter, W.D. (1982) Arch. Microbiol.
132, 297–303
Received 19 September 2003