By using the information given in the article by Ahmed, Ettema, Tjaden, Geerling
ID: 134816 • Letter: B
Question
By using the information given in the article by Ahmed, Ettema, Tjaden, Geerling, Van der oost and Siebers (attached at the end of this problem set),
a) Highlight the main enzymatic reactions involved in the classical ED pathway?
b) Highlight the main reactions of the semi-phosphorylative ED pathway?
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, Universita¨ tsstr.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 3- phosphate)-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. 1To whom correspondence should be addressed (e-mail bettina.siebers@uni-essen.de). 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 13C-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 EMPpathway 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-?-glucosidase. The identity of the ED genes could be confirmed by biochemical characterization of their encoded products after expression in C 2004 Biochemical Society 304 Biochemical Society Transactions (2004) Volume 32, part 2 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-?-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), noATP 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 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 carbohydratemetabolism 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., Scha¨ fer, 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 Scho¨ nheit, P. (1994) Arch. Microbiol. 162, 286–294 6 Selig, M., Xavier, K.B., Santos, H. and Scho¨ nheit, 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 D¨ orr, 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., Do¨ rr, 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 Ko¨ nig, H., Skorko, R., Zillig, W. and Reiter, W.D. (1982) Arch. Microbiol. 132, 297–303 Received 19 September 2003 C 2004 Biochemical Society
Explanation / Answer
Ans a) Main enzymatic reactions in the classical ED pathway is the non-phosphorylative ED pathway. In the classical method or the enzymatic reaction, it is found that by phosphorylating GAPDH catalyses leading to the conversion of GAP to 1,3 – diphosphoglycerate. The finding or the main enzymatic reaction in this form of classical ED pathway is the utilization of the reversible EMP pathway and the non-phosphorylative mode is used for catabolism metabolism. The non-phosphorylative ED pathway leads to formation of two heat labile intermediates which are GAP and dihydroxyacetone phosphate and it can be used for growing at high temperature.
Ans b) The main reactions of the semi-phosphorylative ED pathway and it is found that this pathway is active in hyperthermophiles. This can also be determined due to the presence of KDG kinase which is an semi-phosphorylative form of the pathway. In the main reaction that is associated with the semi-phosphorylative ED pathways, it is found that formation of the extremely heat labile 1,3 – diphosphoglycerate is avoid through single step conversion of the GAP to 3-phosphoglycerate with gelp of GAPN or GAPOR.
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