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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Publications at this Location » Publication #104125


item Gronwald, John

Submitted to: Physiologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/16/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: There is a need to develop alternative, renewable biofuels to reduce dependency on foreign energy sources. The use of soybean oil for production of biodiesel holds promise in this area. Biodiesel from soybeans is a biodegradable fuel that can be used in conventional diesel engines. Compared to conventional diesel fuel, biodiesel is cleaner-burning and has less environmental impact. The availability of extra high-oil soybeans would be desirable for the production of biodiesel. Acetyl-CoA carboxylase (ACCase) is a protein in soybean seeds that is believed to catalyze the rate-limiting step in the synthesis of fatty acids that are used to make oil. It is very likely that increasing the activity of this enzyme would increase oil content of soybeans. We have been conducting research to determine what factors regulate the activity of ACCase in developing soybean seeds. We found that soybean ACCase is activated by iron. This activation is the result of the binding of iron to one component of the ACCase protein. This is the first report that iron may play an important role in regulating the activity of ACCase in plants. The knowledge generated by this research will be useful in the development of extra high- oil producing soybean varieties that would be advantageous for the production of biodiesel.

Technical Abstract: Multi-subunit acetyl-coenzyme A carboxylase (MS-ACCase) isolated from soybean chloroplasts is an unstable enzyme that loses activity following chloroplast lysis. We found that incubating the chloroplast stromal fraction under anaerobic conditions or in the presence of 5 mM FeSO4 stimulated ACCase (acetyl-CoA conversion to malonyl-CoA) and carboxyltransferase (malonyl-CoA conversion to acetyl-CoA) activity. Fe- activation was associated with 59Fe binding to a stromal protein fraction. ACCase and carboxyltransferase activities measured in the stromal protein fraction containing bound 59Fe were 2-fold and 6-fold greater, respectively, than the control (stromal fraction not pretreated with FeSO4). Superose 6 gel filtration chromatography indicated 59Fe comigrated with a stromal protein of approximately 180 kD that exhibited carboxyltransferase activity but lacked ACCase activity. Anion exchange chromatography yielded an apparently homogenous protein peak containing carboxyltransferase activity and protein-bound 59Fe. Denaturing gels indicated the 180 kD protein was composed of a 56 kD subunit that was bound by an antibody raised against a synthetic beta-carboxyltransferase peptide. Incubation of the purified carboxyltransferase with increasing concentrations of iron resulted in increased initial velocities that fit well to a single rectangular three parameter hyperbola (v = vo + Vmax[FeSO4] / Km + [FeSO4]). UV/Vis spectroscopy of the purified fraction before and after iron incubation yielded spectra consistent with a protein bound metal cluster. These results suggest that the beta- carboxyltransferase subunit of the MS-ACCase of soybean chloroplasts is an iron-containing enzyme, which may in part explain its labile nature.