Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1.
The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53,269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80 degrees C; ammonia-dependent reaction, 90 degrees C).
Jongsareejit
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B
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Rahman
R N
RN
Fujiwara
S
S
Imanaka
T
T
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000201:enzymology; D005970:Glutamate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; C107166:glutamate synthase A; D005970:Glutamate Synthase