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Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.
P A Duncan1, B A White, R I Mackie
1Department of Animal Sciences, University of Illinois, Urbana-Champaign 61801.
Applied and Environmental Microbiology
|December 1, 1992
Summary
Glutamate dehydrogenase from Ruminococcus flavefaciens was purified and characterized, revealing unique properties. This enzyme shows highest activity during ammonia-limited growth, unlike other bacterial GDHs.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Ruminococcus flavefaciens is a key cellulolytic bacterium in the rumen.
- Glutamate dehydrogenase (GDH) plays a crucial role in nitrogen metabolism in microorganisms.
Purpose of the Study:
- To purify and characterize the glutamate dehydrogenase (GDH) from Ruminococcus flavefaciens.
- To investigate the kinetic properties and regulatory mechanisms of R. flavefaciens GDH.
Main Methods:
- Enzyme purification techniques.
- Enzyme activity assays under varying conditions (pH, salt concentration, substrate concentrations).
- Kinetic analysis including Km determination and coenzyme saturation curves.
- N-terminal amino acid sequencing.
Main Results:
- Purified R. flavefaciens GDH is a hexamer (280 kDa) with 48 kDa subunits.
- Optimal activity observed at pH 6.9-7.0 and with 0.5 M KCl.
- Km values for ammonia, alpha-ketoglutarate, and glutamate determined.
- Sigmoidal NADPH saturation indicates positive cooperativity.
- N-terminal sequence shows low homology (39%) with other bacterial GDHs.
- Specific activity is highest during ammonia-limited growth and unaffected by ammonia shock.
Conclusions:
- R. flavefaciens GDH exhibits unique kinetic and regulatory properties compared to other bacterial GDHs.
- The enzyme's activity regulation is adapted to the ammonia availability in the ruminal environment.
- N-terminal analysis suggests potential post-translational modifications.