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A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses
Published on: January 25, 2018
Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus
M K Trower1, F S Sariaslani, D P O'Keefe
1E. I. du Pont de Nemours and Company, Central Research and Development Department, Wilmington, Delaware 19880-0228.
Abstract:
A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.
Insights
Researchers purified cytochrome P-450soy from Streptomyces griseus. This enzyme, essential for metabolizing xenobiotics, was reconstituted and showed catalytic activity with specific substrates.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Cytochrome P-450 enzymes are crucial for metabolizing a wide range of compounds, including xenobiotics.
- Streptomyces griseus is a source of microbial enzymes with potential biotechnological applications.
- Understanding P-450 enzyme structure and function is key to predicting and manipulating metabolic pathways.
Purpose of the Study:
- To purify and characterize a soluble cytochrome P-450 (P-450soy) induced by soybean flour from Streptomyces griseus.
- To investigate the catalytic activity of purified P-450soy when reconstituted with essential redox partners.
- To explore the generation of a stable, active P-450 fragment through in vitro proteolysis.
Main Methods:
- Purification of soluble cytochrome P-450soy from Streptomyces griseus using standard biochemical techniques, achieving 130-fold purification.
- Characterization of the purified enzyme's physical and spectral properties, including molecular weight and visible absorption spectra.
- Reconstitution assays using spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase to assess NADPH-dependent catalytic activity with xenobiotic substrates (precocene II, 7-ethoxycoumarin).
- In vitro proteolysis to generate and isolate a stable, active fragment (P-450soy delta).
Main Results:
- Soluble cytochrome P-450soy was purified to homogeneity as a single polypeptide (MW 47,500) with a ferriprotoporphyrin IX prosthetic group.
- Spectroscopic analysis confirmed P-450soy as a high-spin cytochrome P-450.
- Reconstituted P-450soy demonstrated NADPH-dependent oxidation of precocene II and 7-ethoxycoumarin, indicating xenobiotic metabolism capability.
- Proteolysis yielded a stable and catalytically active P-450 fragment, P-450soy delta.
Conclusions:
- Soybean flour induces a functional cytochrome P-450 (P-450soy) in Streptomyces griseus.
- Purified P-450soy, when reconstituted, effectively catalyzes the oxidation of specific xenobiotic substrates.
- A stable and active P-450 domain (P-450soy delta) can be generated, offering potential for further structural and functional studies.
