Purification and characterization of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Streptomyces rimosus

A purification scheme for 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Streptomyces rimosus is presented. A four-step procedure involving conventional chromatography and FPLC led to a 2700-fold enrichment of the enzyme activity present in crude extracts. Enzyme prepared by this procedure was judged to be 97% homogeneous by densitometry of a Coomassie-blue-stained polyacrylamide gel. A novel, sensitive, coupled assay was used to follow the purification. The Michaelis constants of the purified enzyme are 6.7 microM and 2.6 microM for phospho enol pyruvate and erythrose 4-phosphate, respectively. These values are one to two orders of magnitude lower than the Km values reported for partially purified enzyme from other Streptomycetes. A number of potential metabolic effectors were tested for their ability to inhibit the purified enzyme. Tryptophan was found to be a partial inhibitor and appeared to bind to the enzyme cooperatively.