title: Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and catalytic activity.
abstract: Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes the anti-1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, a precursor for the biosynthesis of aromatic compounds. Enzyme activity relies on reduced FMN, which is thought to donate an electron transiently to the substrate, facilitating C(3)-O bond breakage. The crystal structure of the enzyme with bound EPSP and the flavin cofactor highlighted two invariant serine residues interacting with a bound water molecule that is close to the C(3)-O of EPSP. In this article we present the results of a mutagenesis study where we replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate synthase with alanine, producing two single-mutant proteins (Ser16Ala and Ser127Ala) and a double-mutant protein (Ser16AlaSer127Ala). The residual activity of the Ser127Ala and Ser16Ala single-mutant proteins was found to be six-fold and 70-fold lower, respectively, than that of the wild-type protein. No residual activity was detected for the Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, in contrast to the single-mutant proteins. On the basis of the structure of the enzyme, we propose that Ser16 and Ser127 form part of a proton relay system among the isoalloxazine ring of FMN, histidine 106 and the phosphate group of EPSP that is essential for the formation of the transient intermediate and for substrate turnover.
authors.affiliation:  Institute of Biochemistry, Graz University of Technology, Austria.
authors.forename: Gernot
authors.initials: G
authors.lastname: Rauch
authors.affiliation:  Institute of Biochemistry, Graz University of Technology, Austria.
authors.forename: Heidemarie
authors.initials: H
authors.lastname: Ehammer
authors.affiliation:  Department of Biological Chemistry, John Innes Centre, Norwich, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Bornemann
authors.affiliation:  Institute of Biochemistry, Graz University of Technology, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D002384:Catalysis; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D011522:Protons; D012694:Serine / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D005415:Flavins; D011522:Protons; C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; D012694:Serine; C008173:isoalloxazine; D006639:Histidine; D005486:Flavin Mononucleotide; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases; D000409:Alanine