Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability.
The glutamate dehydrogenase gene from the hyperthermophilic archaeon Pyrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda PL promoter. The P. furiosus glutamate dehydrogenase amounted to 20% of the total E. coli cell protein, and the vast majority consisted of hexamers. Following activation by heat treatment, an enzyme could be purified from E. coli that was indistinguishable from the glutamate dehydrogenase purified from P. furiosus. Hybrid genes, that consisted of the coding regions for the homologous glutamate dehydrogenases from P. furiosus and the mesophilic bacterium Clostridium difficile, were constructed and successfully expressed in E. coli. One of the resulting hybrid proteins, containing the glutamate binding domain of the C. difficile enzyme and the cofactor binding domain of the P. furiosus enzyme, did not show a detectable activity. In contrast, the complementary hybrid containing the P. furiosus glutamate and the C. difficile cofactor binding domain was a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the Clostridium symbiosum enzyme. Compared with the C. difficile glutamate dehydrogenase, the archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation.
Lebbink
J H
JH
Department of Microbiology, Wageningen Agricultural University, Netherlands.
Eggen
R I
RI
Geerling
A C
AC
Consalvi
V
V
Chiaraluce
R
R
Scandurra
R
R
de Vos
W M
WM
D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016360:Clostridioides difficile / Q000201:enzymology / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D015870:Gene Expression / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D006146:Guanidines; D011993:Recombinant Fusion Proteins; D005969:Glutamate Dehydrogenase; D019791:Guanidine