scholarly journals Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

2000 ◽  
Vol 182 (9) ◽  
pp. 2559-2566 ◽  
Author(s):  
Donald E. Ward ◽  
Servé W. M. Kengen ◽  
John van der Oost ◽  
Willem M. de Vos
Keyword(s):  
Molecular Mass ◽  
Alanine Aminotransferase ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Northern Analysis ◽  
Transcriptional Level ◽  
Significant Activity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosusby multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in theP. furiosus genome database. The gene was expressed inEscherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. Thek cat/Km values for alanine and pyruvate formation were 41 and 33 s−1mM−1, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aatgene. In addition, both the aat and gdh(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdhgenes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

scholarly journals Characterization of an Aminoacylase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2001 ◽  
Vol 183 (14) ◽  
pp. 4259-4268 ◽  
Author(s):  
Sherry V. Story ◽  
Amy M. Grunden ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Pyrococcus Furiosus ◽  
Protein A ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Protein Renaturation ◽  
Cell Extracts ◽  
Growth Substrates ◽  
Catalytically Active

ABSTRACT Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-l-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 ± 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl2 (and to 74% by the addition of CoCl2). After reconstitution with ZnCl2, the enzyme contained 2.85 ± 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolarN-acylated l amino acids (Met, Ala, Val, and Leu), as well as N-formyl-l-methionine. The high Km values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity withN-acetyl-l-methionine as the substrate occurred at pH 6.5 and a temperature of 100°C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in theP. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn2+ ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of theP. furiosus aminoacylase (≥50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.

scholarly journals Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

scholarly journals Role of the β1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic Archaeon Pyrococcus furiosus

2006 ◽  
Vol 189 (2) ◽  
pp. 583-590 ◽  
Author(s):  
Lara S. Madding ◽  
Joshua K. Michel ◽  
Keith R. Shockley ◽  
Shannon B. Conners ◽  
Kevin L. Epting ◽  
...  
Keyword(s):  
Thermal Stress ◽  
Pyrococcus Furiosus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Response Shift ◽  
Transcriptional Analysis ◽  
20S Proteasome ◽  
Significant Activity ◽  
Peptide Hydrolysis ◽  
Hyperthermophilic Archaeon

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one α protein (PF1571) and two β proteins (β1-PF1404 and β2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105°C) showed that the β1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the β2/β1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant α+β1+β2 CP assembled at 105°C was more thermostable than either the α+β1+β2 version assembled at 90°C or the α+β2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the α and β1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the β1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that β subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

scholarly journals Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

1998 ◽  
Vol 180 (18) ◽  
pp. 4781-4789 ◽  
Author(s):  
Mousumi Ghosh ◽  
Amy M. Grunden ◽  
Dianne M. Dunn ◽  
Robert Weiss ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Metal Ion ◽  
Pyrococcus Furiosus ◽  
Cobalt Atom ◽  
Significant Similarity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd , 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, theKm values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greaterV max value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

scholarly journals Phosphoenolpyruvate Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2001 ◽  
Vol 183 (2) ◽  
pp. 709-715 ◽  
Author(s):  
Andrea M. Hutchins ◽  
James F. Holden ◽  
Michael W. W. Adams
Keyword(s):  
Molecular Mass ◽  
Functional Role ◽  
Pyrococcus Furiosus ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Kinetic Properties ◽  
Additional Function ◽  
Hyperthermophilic Archaeon ◽  
A Subunit

ABSTRACT Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690 ± 20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large (∼1.6 MDa) complex that is not active. The apparentKm values and catalytic efficiencies for the substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and 1.43 × 104 mM−1 · s−1 and 0.39 mM and 3.40 × 103mM−1 · s−1, respectively. Maximal activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k cat/Km = 32 (mM · s)−1]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration (∼5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

scholarly journals Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes

1999 ◽  
Vol 181 (11) ◽  
pp. 3358-3367 ◽  
Author(s):  
Karina B. Xavier ◽  
Ralf Peist ◽  
Marina Kossmann ◽  
Winfried Boos ◽  
Helena Santos
Keyword(s):  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Maximal Activity ◽  
Consensus Binding Site ◽  
Calculated Molecular Mass ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
Key Enzymes ◽  
Maltose Metabolism ◽  
Cell Extracts

ABSTRACT Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected inT. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, ap-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. AKm of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacteriumThermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.

scholarly journals Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

1998 ◽  
Vol 180 (17) ◽  
pp. 4591-4595 ◽  
Author(s):  
Zhongqi He ◽  
John K. Davis ◽  
Jim C. Spain
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Pseudomonas Pseudoalcaligenes ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts ◽  
High Degree

ABSTRACT 2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.

scholarly journals An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

1999 ◽  
Vol 181 (4) ◽  
pp. 1163-1170 ◽  
Author(s):  
Kesen Ma ◽  
Michael W. W. Adams
Keyword(s):  
Alcohol Dehydrogenase ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Physiological Role ◽  
Resonance Spectroscopy ◽  
Hyperthermophilic Archaeon ◽  
Subunit Molecular Weight ◽  
Cell Extracts ◽  
Oxygen Sensitive

ABSTRACT Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO2, and H2, together with minor amounts of ethanol. The organism also generates H2S in the presence of elemental sulfur (S0). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S0. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (∼1.0 g-atom/subunit) and zinc (∼1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent Km s (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.

scholarly journals Cold Shock of a Hyperthermophilic Archaeon: Pyrococcus furiosus Exhibits Multiple Responses to a Suboptimal Growth Temperature with a Key Role for Membrane-Bound Glycoproteins

2005 ◽  
Vol 187 (1) ◽  
pp. 336-348 ◽  
Author(s):  
Michael V. Weinberg ◽  
Gerrit J. Schut ◽  
Scott Brehm ◽  
Susmita Datta ◽  
Michael W. W. Adams
Keyword(s):  
Growth Temperature ◽  
Cold Shock ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Optimal Growth ◽  
Cold Shock Protein ◽  
Hyperthermophilic Archaea ◽  
Lag Phase ◽  
Cold Response ◽  
Hyperthermophilic Archaeon

ABSTRACT The hyperthermophilic archaeon, Pyrococcus furiosus, was grown on maltose near its optimal growth temperature, 95°C, and at the lower end of the temperature range for significant growth, 72°C. In addition, cultures were shocked by rapidly dropping the temperature from 95 to 72°C. This resulted in a 5-h lag phase, during which time little growth occurred. Transcriptional analyses using whole-genome DNA microarrays representing 2,065 open reading frames (ORFs) in the P. furiosus genome showed that cells undergo three very different responses at 72°C: an early shock (1 to 2 h), a late shock (5 h), and an adapted response (occurring after many generations at 72°C). Each response involved the up-regulation in the expression of more than 30 ORFs unique to that response. These included proteins involved in translation, solute transport, amino acid biosynthesis, and tungsten and intermediary carbon metabolism, as well as numerous conserved-hypothetical and/or membrane-associated proteins. Two major membrane proteins were evident after one-dimensional sodium dodecyl sulfate-gel analysis of cold-adapted cells, and staining revealed them to be glycoproteins. Their cold-induced expression evident from the DNA microarray analysis was confirmed by quantitative PCR. Termed CipA (PF0190) and CipB (PF1408), both appear to be solute-binding proteins. While the archaea do not contain members of the bacterial cold shock protein (Csp) family, they all contain homologs of CipA and CipB. These proteins are also related phylogenetically to some cold-responsive genes recently identified in certain bacteria. The Cip proteins may represent a general prokaryotic-type cold response mechanism that is present even in hyperthermophilic archaea.

scholarly journals Identification and characterization of Thermoplasma acidophilum glyceraldehyde dehydrogenase: a new class of NADP+-specific aldehyde dehydrogenase

2006 ◽  
Vol 397 (1) ◽  
pp. 131-138 ◽  
Author(s):  
Jin Hwa Jung ◽  
Sun Bok Lee
Keyword(s):  
Molecular Mass ◽  
Aldehyde Dehydrogenase ◽  
Aromatic Aldehydes ◽  
Thermoplasma Acidophilum ◽  
Genome Database ◽  
New Class ◽  
Cell Extracts ◽  
Sds Page ◽  
Aldehyde Dehydrogenase Superfamily ◽  
Inhibition Studies

Thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus solfataricus are known to metabolize D-glucose via the nED (non-phosphorylated Entner–Doudoroff) pathway. In the present study, we identified and characterized a glyceraldehyde dehydrogenase involved in the downstream portion of the nED pathway. This glyceraldehyde dehydrogenase was purified from T. acidophilum cell extracts by sequential chromatography on DEAE-Sepharose, Q-Sepharose, Phenyl-Sepharose and Affi-Gel Blue columns. SDS/PAGE of the purified enzyme showed a molecular mass of approx. 53 kDa, whereas the molecular mass of the native protein was 215 kDa, indicating that glyceraldehyde dehydrogenase is a tetrameric protein. By MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS) peptide fingerprinting of the purified protein, it was found that the gene product of Ta0809 in the T. acidophilum genome database corresponds to the purified glyceraldehyde dehydrogenase. The native enzyme showed the highest activity towards glyceraldehyde, but no activity towards aliphatic or aromatic aldehydes, and no activity when NAD+ was substituted for NADP+. Analysis of the amino acid sequence and enzyme inhibition studies indicated that this glyceraldehyde dehydrogenase belongs to the ALDH (aldehyde dehydrogenase) superfamily. BLAST searches showed that homologues of the Ta0809 protein are not present in the Sulfolobus genome. Possible differences between T. acidophilum (Euryarchaeota) and S. solfataricus (Crenarchaeaota) in terms of the glycolytic pathway are thus expected.

Sign in / Sign up

Export Citation Format

Share Document