Glucose metabolism in the extreme thermoacidophilic archaebacterium <i>Sulfolobus solfataricus</i>

Rosa, Mario De; Gambacorta, Agata; Nicolaus, Barbara; Giardina, Paola; Poerio, Elia; Buonocore, Vincenzo

doi:10.1042/bj2240407

Cited by 162 publications

(127 citation statements)

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“…Initial biochemical studies in Archaea revealed the presence of the non-phosphorylative (np) ED pathway in (hyper)thermophiles (De Rosa et al 1984;Siebers and Hensel 1993;Siebers et al 1997;Selig et al 1997) and the semi-phosphorylative (sp) ED pathway in halophilic archaea (Johnsen et al 2001;Tomlinson et al 1974). However, recent combinations of genomics-based and biochemical analyses demonstrated the presence of the branched ED pathway in all Archaea that utilize the ED pathway (Siebers et al 2004;Ahmed et al 2005;Jung and Lee 2005;Kehrer et al 2007).…”

Section: Introductionmentioning

confidence: 99%

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The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

et al. 2007

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Archaea utilize a branched modification of the classical Entner-Doudoroff (ED) pathway for sugar degradation. The semi-phosphorylative branch merges at the level of glyceraldehyde 3-phosphate (GAP) with the lower common shunt of the Emden-Meyerhof-Parnas pathway. In Sulfolobus solfataricus two different GAP converting enzymes-classical phosphorylating GAP dehydrogenase (GAPDH) and the non-phosphorylating GAPDH (GAPN)-were identified. In Sulfolobales the GAPN encoding gene is found adjacent to the ED gene cluster suggesting a function in the regulation of the semi-phosphorylative ED branch. The biochemical characterization of the recombinant GAPN of S. solfataricus revealed that-like the well-characterized GAPN from Thermoproteus tenax-the enzyme of S. solfataricus exhibits allosteric properties. However, both enzymes show some unexpected differences in co-substrate specificity as well as regulatory fine-tuning, which seem to reflect an adaptation to the different lifestyles of both organisms. Phylogenetic analyses and database searches in Archaea indicated a preferred distribution of GAPN (and/or GAP oxidoreductase) in hyperthermophilic Archaea supporting the previously suggested role of GAPN in metabolic thermoadaptation. This work suggests an important role of GAPN in the regulation of carbon degradation via modifications of the EMP and the branched ED pathway in hyperthermophilic Archaea.Keywords Glyceraldehyde-3-phosphate Á Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase Á GAPN Á Aldehyde dehydrogenase superfamily Á Branched Entner-Doudoroff pathway Á Carbohydrate metabolism Á Archaea Abbreviations EDEntner-Doudoroff sp Semi-phosphorylative np Non-phosphorylative EMP Embden-Meyerhof-Parnas G1P

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Section: Introductionmentioning

confidence: 99%

“…The subsequent phosphorylation by glycerate kinase (MOFRL family) results in the formation of 2-phosphoglycerate (Kehrer et al 2007;. Finally, pyruvate is formed by the combined action of enolase and pyruvate kinase (De Rosa et al 1984;Selig et al 1997).…”

Section: Introductionmentioning

confidence: 99%

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

et al. 2007

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“…Sulfolobus solfataricus, strain MT-4, was isolated from an acidic hot spring in Agnano (Naples) and grown as reported [34]. The homogenate preparation was carried out according to De Rosa et al [34] using as buffer 10 mM Tris/HCl pH 8.…”

Section: Cell Culture and Homogenate Preparationmentioning

confidence: 99%

“…The homogenate preparation was carried out according to De Rosa et al [34] using as buffer 10 mM Tris/HCl pH 8. The homogenate was then added with streptomycin sulfate (25 ml freshly prepared 12% solution) and centrifuged at 15000 x g for 30 min (high-speed supernatant).…”

Section: Cell Culture and Homogenate Preparationmentioning

confidence: 99%

S‐Adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus.

Porcelli¹,

Cacciapuoti²,

Cartenı́-Farina³

et al. 1988

European Journal of Biochemistry

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Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfuturicus, a thermophilic archaebacterium optimally growing at 87 "C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B.The A and B isoforms follow Michaelis-Menten kinetics with apparent K , values of 10 pM nd 20 pM for L-methionine and of 50 pM and 150 pM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non-competitive inhibitor (Ki = 50 pM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 pM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents.The optimum temperature is 90°C and no significant loss of activity is observable after incubation of the two isoforms at 100 "C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75 "C.The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.The metabolic importance of S-adenosylmethionine (AdoMet) in both eukaryotic and prokaryotic organisms is well known. Besides its major roles as methyl and aminopropyl donor, the sulfonium compound functions either as substrate or as modulator of a number of enzymatic systems [l -51. In contrast, only few data are available in the literature on the metabolism of AdoMet in archaebacteria [6, 71, which represent the third line of evolution, completely distinct from the eukaryotic and eubacterial lines [8].Recently, in halophilic and methanogenic archaebacteria, methylated DNA has been identified [9], but no data have been reported so far on the molecular features of the enzymes involved in the biosynthesis and metabolism of AdoMet in such microorganisms.In Sulfolobus solfuturicus, an extreme thermoacidophilic archaebacterium, optimally growing at 8 7 T , the role of AdoMet as methyl donor to proteins and as precursor of the symmetrical polyamines has been established [lo, 111.In eubacteria as well as in eukaryotes the synthesis of AdoMet from methionine and ATP is catalyzed by Ado- Met synthetase (ATP:L-methionine S-adenosyltransferase) through an unusual reaction in which the energy-rich sulfonium compound is formed by the complete dephosphorylation of ATP [12]. Pyrophosphate originating from the a and fi phosphates of ATP, and orthophosphate originating from the y phosphate, together represent the other products of the enzymatic reaction [12]. This finding implies that inorganic tripolyphosphate could be an enzyme-bound intermediate which is cleaved by a trip...

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“…solfataricus, strain MT-4, was isolated from an acidic hot spring in Agnano (Naples) and grown as reported [36]. The homogenate preparation was carried out according to De Rosa et al [36] using as buffer 10 mM Tris/HCl pH 8. The homogenate was then added with streptomicin sulfate (25 ml freshly prepared 12% solution) and centrifuged at 15000 x g for 30 min.…”

Section: Cell Culture and Homogenate Preparationmentioning

confidence: 99%

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium

Cacciapuoti¹,

Porcelli²,

Cartenı́-Farina³

et al. 1986

Eur J Biochem

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The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfaturicus, an extreme thermophilic archaebacterium optimally growing at 87 "C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90°C and no loss of activity is observable even after exposure of the purified enzyme to 100°C for 1 h. No reducing agents are required for enzymatic activity.Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane ( K , = 1675 pM), putrescine (K, = 3850 pM), sym-norspermidine (K, = 954 pM) and spermidine (K, = 1539 pM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (K, = 7.9 pM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 pM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-l,8-diaminooctane are the most active inhibitors.Propylamine transferases represent a group of enzymes catalyzing the transfer of the propylamine moiety from S-adenosyl-(5')-3-methylthiopropylamine (decarboxy-AdoMet) to appropriate amine acceptors [l -41. The reaction involves a nucleophilic attack on the methylene C-3 adjacent to the positively charged sulfur of decarboxy-AdoMet [5]. This class of enzymes is distributed in both prokaryotic and eukaryotic organisms. The recent development of rapid and sensitive methods for polyamine analysis [6 -81 and the utilization of affinity chromatography [9, 101 has made possible their purification and characterization : indeed spermidine Correspondence to V. Zappia, Istituto di Biochimica delle Macromolecole, I Facolta di Medicina e Chirurgia, Universita di Napoli, Via Costantinopoli 16, 1-80138, Napoli, ItalyAbbreviations. Decarboxy-AdoMet, S-adenosyl(S')-3-methylthiopropylamine; sym-norspermidine, 1,7-diamino-4-aza-eptane; symnorspermine, 1,l l-diamino-4,8-diaza-undecane; sym-homospermidine, 1,9-diamin0-5-aza-nonane; caldopentamine, 1,15-diamino- synthase has been purified from ra...

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Glucose metabolism in the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus

Cited by 162 publications

References 15 publications

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

S‐Adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus.

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium

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