Fig. 3: Enzyme assays of 6-phosphogluconate dehydratase (EDD) and dihydroxyacid dehydratase (DHAD).

Soybean (Gm) DHAD and E. coli (Ec) EDD and DHAD were expressed in E. coli, and purified proteins were used individually or in combination to evaluate respective activity of EDD and DHAD. a Coupled ED assays using 6-phosphogluconate as the substrate demonstrate that EDD, but not DHAD, provides KDPG which can be further converted to GAP and pyruvate by KDPG aldolase (EDA) of plant or bacterial origin. Bacterial proteins and product chromatograms are shown in brown; plants in green. b DHAD assay using 2,3-dihydroxyisovalerate (DIV) to produce 2-ketoisovalerate (KIV). Both E. coli (Ec) and soybean DHAD converted DIV to KIV, while E. coli EDD did not. c DHAD assay using crude protein (CP) extract from soybean and DIV supplied as substrate displaying detectable DHAD activity in soybean leaf extracts. d The same soybean crude protein extract supplied with 6-phosphogluconate did not yield the expected EDD dehydration product, 2-keto-3-deoxy-6-phosphogluconate (KDPG). Each reaction was repeated at least once with identical results.