Abstract
Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover 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 is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.
Highlights
Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis
Because binding of oxidized FMN to the N. crassa chorismate synthase can be assumed to be a prerequisite for its reduction by NADPH, it is conceivable that formation of a ternary complex of enzyme, FMN, and enolpyruvylshikimate 3-phosphate (EPSP) is required before reduction can occur
Expression and Purification of N. crassa Chorismate Synthase—Heterologous expression of NcCS in conventional E. coli BL21 (DE3) cells resulted in low yields of protein because of the usage of rare codons in the N. crassa cDNA
Summary
Reagents—All chemicals were of the highest grade available and obtained from Sigma or Fluka (Buchs, Switzerland). To remove chorismate synthase from the protein preparation, the dialyzed sample was loaded onto a cellulose phosphate column (2.5 ϫ 16 cm) prepared according to the manufacturer’s instructions and subsequently equilibrated with buffer B. The precipitate from 50% ammonium sulfate saturation, which contained NcCS, was collected by centrifugation and dissolved in buffer A, dialyzed against the same buffer overnight, and subjected to anion exchange chromatography on a DEAE-Sephacel column (2.5 ϫ 16 cm). The reaction buffer contained 0.1 M potassium phosphate, pH 7.6, 4 mM MgSO4, 10 mM glutamine, 0.03 M ammonium sulfate, 1 mM dithiothreitol, 10 M FMN, 80 M EPSP, and 50 picokatal of recombinant anthranilate synthase component I from E. coli.
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