Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.
The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.
Adul Rahman
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Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Jongsareejit
B
B
Fujiwara
S
S
Imanaka
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase