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Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes theanti-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 com-pounds. | ễFEBS Journal Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and catalytic activity Gernot Rauch1 Heidemarie Ehammer1 Stephen Bornemann2 and Peter Macheroux1 1 Institute of Biochemistry Graz University of Technology Austria 2 Department of BiologicalChemistry John Innes Centre Norwich UK Keywords enzyme mechanism elimination flavin shikimate pathway site-directed mutagenesis Correspondence P. Macheroux Institute of Biochemistry Graz University of Technology Petersgasse 12 II A-8010 Graz Austria Fax 43-316-873 6952 Tel 43-316-8736450 E-mail peter.macheroux@tugraz.at Received 6 December 2007 revised 15 January 2008 accepted 21 January 2008 doi 10.1111 j.1742-4658.2008.06305.x 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 Ser16Ala-Ser127Ala . The residual activity of the Ser127Ala and Ser16Ala singlemutant 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 .
