Related Experiment Videos
Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians
L Humanes1, A López-Ruiz, M T Merino
1Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Spain.
Applied and Environmental Microbiology
|September 18, 1997
Summary
Rhodococcus fascians limonoate dehydrogenase was purified and characterized. Limonoate A-ring lactone is confirmed as the physiological substrate, with higher enzyme activity observed when grown on limonoate.
Area of Science:
- Biochemistry
- Enzymology
- Microbial Physiology
Background:
- Limonoate dehydrogenase (LDH) plays a role in limonoid metabolism.
- Understanding LDH's substrate specificity and regulation is crucial for microbial biochemistry.
- Previous studies suggested limonoate A-ring lactone as the physiological substrate.
Purpose of the Study:
- To purify and characterize limonoate dehydrogenase from Rhodococcus fascians.
- To determine the enzyme's substrate preference and kinetic properties.
- To investigate the regulation of LDH expression in response to carbon sources.
Main Methods:
- Purification of LDH using ion-exchange, hydrophobic, and affinity chromatography.
- Determination of enzyme molecular mass, subunit composition, and isoelectric point.
- NH2-terminal amino acid sequencing and kinetic assays with different limonoids.
Main Results:
- Homogeneous LDH (128,000 Da, tetrameric subunits) was obtained.
- LDH exhibited higher catalytic rates and affinity for limonoate A-ring lactone than disodium limonoate.
- Enzyme activity and protein levels increased in limonoate-grown cultures, suggesting transcriptional regulation.
Conclusions:
- Limonoate A-ring lactone is the physiological substrate for Rhodococcus fascians LDH.
- Enzyme expression is induced by limonoate, indicating metabolic regulation.
- Purified LDH provides a basis for further structural and functional studies.