Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs.
The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47,300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100 degrees C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.
Rahman
R N
RN
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Fujiwara
S
S
Takagi
M
M
Imanaka
T
T
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D019766:Genes, Archaeal / Q000235:genetics*; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase