| Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1. | |
| | |
MedLine Citation:
|
PMID: 9202379 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
|
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). |
| | |
Authors:
|
B Jongsareejit; R N Rahman; S Fujiwara; T Imanaka |
Related Documents
:
|
16385059 - Identification and characterization of a novel bacterial sulfite oxidase with no heme b... 1856159 - Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase ... 17986789 - Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic... 15383719 - Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from pseudo... 9644249 - Molecular cloning, expression, and site-directed mutagenesis of inorganic pyrophosphata... 7717969 - Evidence for a general role for non-catalytic thermostabilizing domains in xylanases fr... 7873779 - Characterization of avrpphe, a gene for cultivar-specific avirulence from pseudomonas s... 6304649 - Functional analysis of a herpes simplex virus type 1 promoter: identification of far-up... 23273849 - Expression of sulfatases in rhodopirellula baltica and the diversity of sulfatases in t... |
Publication Detail:
|
Type: Journal Article |
Journal Detail:
|
Title: Molecular & general genetics : MGG Volume: 254 ISSN: 0026-8925 ISO Abbreviation: Mol. Gen. Genet. Publication Date: 1997 May |
Date Detail:
|
Created Date: 1997-08-15 Completed Date: 1997-08-15 Revised Date: 2004-07-09 |
Medline Journal Info:
|
Nlm Unique ID: 0125036 Medline TA: Mol Gen Genet Country: GERMANY |
Other Details:
|
Languages: eng Pagination: 635-42 Citation Subset: IM |
Affiliation:
|
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan. |
| Data Bank Information | |
Bank Name/Acc. No.:
|
GENBANK/D86223 |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
Amino Acid Sequence Archaea / enzymology*, genetics Base Sequence Chromosomes, Bacterial Cloning, Molecular Eukaryotic Cells / enzymology Glutamate Synthase / genetics*, isolation & purification, metabolism* Molecular Sequence Data Recombinant Proteins / genetics, isolation & purification, metabolism Sequence Analysis Sequence Homology, Amino Acid |
| Chemical | |
Reg. No./Substance:
|
0/Recombinant Proteins; EC 1.4.1.-/glutamate synthase A; EC 1.4.1.13/Glutamate Synthase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: A trans-acting modifier causing extensive overexpression of genes in Drosophila melanogaster.
Next Document: Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI, active i...