Characterization of aromatic and aliphatic 2-ketoacid oxidoreductases from hyperthermophilic archaea

1993 ◽  
Vol 51 (1-2) ◽  
pp. 459 ◽  
Author(s):  
Xuhong Mai ◽  
Michael W.W. Adams
Keyword(s):  
Hyperthermophilic Archaea

Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis

2021 ◽  
Author(s):  
Ryushi Kawakami ◽  
Chinatsu Kinoshita ◽  
Tomoki Kawase ◽  
Mikio Sato ◽  
Junji Hayashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Enzyme Stability ◽  
Hyperthermophilic Archaea ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
Aminobutyrate Aminotransferase ◽  
Chaperone Protein ◽  
Amino Acid Racemase

Abstract The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

scholarly journals Physiological and Genomic Characterization of a Hyperthermophilic Archaeon Archaeoglobus neptunius sp. nov. Isolated From a Deep-Sea Hydrothermal Vent Warrants the Reclassification of the Genus Archaeoglobus

2021 ◽  
Vol 12 ◽  
Author(s):  
Galina Slobodkina ◽  
Maxime Allioux ◽  
Alexander Merkel ◽  
Marie-Anne Cambon-Bonavita ◽  
Karine Alain ◽  
...  
Keyword(s):  
Deep Sea ◽  
Hydrothermal Vent ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Hyperthermophilic Archaea ◽  
The Novel ◽  
Flagellar Motility ◽  
Isolation And Characterization ◽  
Mid Atlantic Ridge

Hyperthermophilic archaea of the genus Archaeoglobus are the subject of many fundamental and biotechnological researches. Despite their significance, the class Archaeoglobi is currently represented by only eight species obtained as axenic cultures and taxonomically characterized. Here, we report the isolation and characterization of a new species of Archaeoglobus from a deep-sea hydrothermal vent (Mid-Atlantic Ridge, TAG) for which the name Archaeoglobus neptunius sp. nov. is proposed. The type strain is SE56T (=DSM 110954T = VKM B-3474T). The cells of the novel isolate are motile irregular cocci growing at 50–85°C, pH 5.5–7.5, and NaCl concentrations of 1.5–4.5% (w/v). Strain SE56T grows lithoautotrophically with H2 as an electron donor, sulfite or thiosulfate as an electron acceptor, and CO2/HCO3− as a carbon source. It is also capable of chemoorganotrophic growth by reduction of sulfate, sulfite, or thiosulfate. The genome of the new isolate consists of a 2,115,826 bp chromosome with an overall G + C content of 46.0 mol%. The whole-genome annotation confirms the key metabolic features of the novel isolate demonstrated experimentally. Genome contains a complete set of genes involved in CO2 fixation via reductive acetyl-CoA pathway, gluconeogenesis, hydrogen and fatty acids oxidation, sulfate reduction, and flagellar motility. The phylogenomic reconstruction based on 122 conserved single-copy archaeal proteins supported by average nucleotide identity (ANI), average amino acid identity (AAI), and alignment fraction (AF) values, indicates a polyphyletic origin of the species currently included into the genus Archaeoglobus, warranting its reclassification.

scholarly journals Characterization of an archaeal malic enzyme from the hyperthermophilic archaeonThermococcus kodakaraensisKOD1

Archaea ◽  
2005 ◽  
Vol 1 (5) ◽  
pp. 293-301 ◽  
Author(s):  
Wakao Fukuda ◽  
Yulia Sari Ismail ◽  
Toshiaki Fukui ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Enzyme Activity ◽  
Malic Enzyme ◽  
Hyperthermophilic Archaea ◽  
Oxidative Decarboxylation ◽  
Limited Information ◽  
Gene Encoding ◽  
Malic Enzyme Activity ◽  
Reductive Carboxylation ◽  
Reaction Direction

Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeonT hermococcus kodakaraensisKOD1 (Tk-Mae). The amino acid sequence ofTk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure ofTk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km) ofTk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encodingTk-Mae and intracellular malic enzyme activity inT. kodakaraensiswere constitutively weak, regardless of the growth substrates. Possible roles ofTk-Mae are discussed based on these results and the metabolic pathways ofT. kodakaraensisdeduced from the genome sequence.

scholarly journals Cloning and Expression of the Inositol Monophosphatase Gene from Methanococcus jannaschii and Characterization of the Enzyme

1998 ◽  
Vol 64 (7) ◽  
pp. 2609-2615 ◽  
Author(s):  
Liangjing Chen ◽  
Mary F. Roberts
Keyword(s):  
Gene Product ◽  
Heat Stability ◽  
Cloning And Expression ◽  
Hyperthermophilic Archaea ◽  
Methanococcus Jannaschii ◽  
Inositol Monophosphatase ◽  
E Coli ◽  
Low Concentrations ◽  
High Concentrations

ABSTRACT Inositol monophosphatase (EC 3.1.3.25 ) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1′-phosphate, an osmolyte found in hyperthermophilic archaea. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (Km = 0.091 ± 0.016 mM;V max = 9.3 ± 0.45 μmol of Pi min−1 mg of protein−1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans

Extremophiles ◽  
2000 ◽  
Vol 4 (4) ◽  
pp. 215-225 ◽  
Author(s):  
Marie-Anne Cambon-Bonavita ◽  
Philippe Schmitt ◽  
Montserrat Zieger ◽  
Jean-Michel Flaman ◽  
Françoise Lesongeur ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Hyperthermophilic Archaea ◽  
Dna Polymerase I ◽  
Polymerase I

Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp

1999 ◽  
Vol 65 (5) ◽  
pp. 1991-1997 ◽  
Author(s):  
Yoshihisa Tachibana ◽  
Akiko Kuramura ◽  
Naoki Shirasaka ◽  
Yuji Suzuki ◽  
Tomoko Yamamoto ◽  
...  
Keyword(s):  
Hot Spring ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
16S Rrna Gene Sequencing ◽  
Hyperthermophilic Archaea ◽  
Rrna Gene ◽  
Optimum Ph ◽  
Rrna Gene Sequencing ◽  
Cyclomaltodextrin Glucanotransferase

ABSTRACT The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genusThermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.

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