Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa - Evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate gro up and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yi eld chorismate, a central building block in aromatic amino acid biosynthesi s. The enzyme has an absolute requirement for reduced FMN, which in the cas e of the fungal chorismate synthases is supplied by an intrinsic FMN: NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic a ctivity. Therefore, these fungal enzymes have been termed "bifunctional." W e have cloned chorismate synthase from the common bread mold Neurospora cra ssa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant IV. crassa ch orismate synthase has a diaphorase activity, i.e. it catalyzes the reductio n of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a re duced Ravin intermediate was observed under single and multiple turnover co nditions with spectral features similar to those reported for monofunctiona l chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnov er experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases i s embedded in the general protein structure and a special NADPH binding dom ain is not required to generate the intrinsic oxidoreductase activity.