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 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.
Kitzing
K
K
Eidgenössische Technische Hochschule Zürich, Institut für Pflanzenwissenschaften, Universitätstr. 2, CH-8092 Zürich, Switzerland.
Macheroux
P
P
Amrhein
N
N
D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
D018076:DNA, Complementary; C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; D009249:NADP; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases; D010100:Oxygen