Mutagenic analysis of an invariant aspartate residue in chorismate synthase supports its role as an active site base.
Chorismate synthase catalyzes the anti-1,4-elimination of the 3-phosphate and the C(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, the final product of the common shikimate pathway and a precursor for the biosynthesis of aromatic compounds. The enzyme has an absolute requirement for reduced FMN, which is thought to facilitate cleavage of C-O bonds by transiently donating an electron to the substrate. The crystal structure of the enzyme revealed that EPSP is bound near the flavin isoalloxazine ring with several invariant amino acid residues in contact with the substrate and/or cofactor. Here, we report the results of a mutagenesis study in which an invariant aspartate residue at position 367 of the Neurospora crassa chorismate synthase was replaced with alanine and asparagine. Both single mutant proteins (Asp367Ala and Asp367Asn) were comparable to the wild-type enzyme with respect to substrate and cofactor binding, indicating that Asp367 is not required for binding of either the flavin or the substrate. In sharp contrast to these results, the activity of both single mutant proteins was found to be 620 and 310 times lower for the Asp367Ala and Asp367Asn mutant proteins, respectively. This finding provides strong evidence that the carboxylate group of Asp367 plays a major role during the catalytic reaction. On the basis of the structure of the enzyme, our data provide the first experimental evidence that the carboxylate group of aspartate 367 participates in the deprotonation of N(5) of the reduced flavin cofactor, which in turn abstracts the C(6proR) hydrogen yielding chorismate as the product.
Rauch
Gernot
G
Institute of Biochemistry, Graz University of Technology, Graz, Austria.
Ehammer
Heidemarie
H
Bornemann
Stephen
S
Macheroux
Peter
P
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002827:Chorismic Acid / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009492:Neurospora crassa / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D001224:Aspartic Acid; D005486:Flavin Mononucleotide; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases; D002827:Chorismic Acid