title: Fluorimetric analysis of the binding characteristics of 5-enolpyruvylshikimate-3-phosphate synthase with substrates in Dunaliella salina.
abstract: A general model of the catalytic mechanism for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) has already been proposed. But whether shikimate-3-phosphate (S3P) alone can cause EPSPs' conformation changes, and whether the binding site of phosphoenolpyruvate (PEP) and glyphosate is the same are still in debate. In this paper, DsaroA gene amplified and cloned from Dunaliella salina (our laboratory's early study) was used for DsEPSPs expression and purification. Then the DsEPSP conformation changes as it bind with different substrates were detected by fluorimetry. The results show that we obtained the DsEPSPs by prokaryotic expression and purification, and the S3P binding with DsEPSPs alone cannot cause DsEPSPs to form "close" conformation directly. However, when S3P exits, DsEPSPs did have a trend to change to the "close" conformation. Then the "close" conformation can be formed completely with the addition of phosphoenolpyruvate (PEP) or glyphosate. The inorganic phosphorus can help S3P to induce two domains of DsEPSPs to form "close" conformation. Besides, when DsEPSPs binds with S3P, in 295 nm, only the intensity of emission peak decreases, however, in 280 nm, not only the peak intensity reduces but also the blue-shift phenomenon takes place. The reason for blue-shift phenomenon was the distribution of aromatic amino acids in EPSPs. EPSPs is a good target for novel antibiotics and herbicides, because of shikimic acid pathway is only present in plants and microorganisms, completely absent in mammals, fish, birds, reptiles, and insects. The results demonstrate that the binding of substrates to EPSPs causes a conformational change from an open form to a closed form, that might be important for designing of novel antimicrobial and herbicidal agents that block closure of the enzyme.
authors.affiliation: Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu, Sichuan, P. R. China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Cao
authors.forename: Hui
authors.initials: H
authors.lastname: Xu
authors.forename: Li
authors.initials: L
authors.lastname: Xie
authors.forename: Yi
authors.initials: Y
authors.lastname: Yi
authors.forename: Yingpeng
authors.initials: Y
authors.lastname: Yu
authors.forename: Shunli
authors.initials: S
authors.lastname: Feng
authors.forename: Dairong
authors.initials: D
authors.lastname: Qiao
authors.forename: Yi
authors.initials: Y
authors.lastname: Cao
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D005470:Fluorometry; D015870:Gene Expression; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D008958:Models, Molecular; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D016823:Volvocida / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D005998:Glycine; D010728:Phosphoenolpyruvate; D011994:Recombinant Proteins; D012765:Shikimic Acid; D000097797:Glyphosate; C042786:shikimic acid-3-phosphate
keywords: 5-Enolpyruvylshikimate-3-phosphate synthase; Dunaliella salina; Fluorescence spectrum; Glyphosate; Inorganic phosphate anion