title: The biochemical properties of the twoArabidopsis thalianaisochorismate synthases.
abstract: The important plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) regulates several key plant responses including, most notably, defence against pathogens. A key enzyme for SA biosynthesis is isochorismate synthase (ICS), which converts chorismate into isochorismate, and for which there are two genes inArabidopsis thalianaOne (AtICS1) has been shown to be required for increased SA biosynthesis in response to pathogens and its expression can be stimulated throughout the leaf by virus infection and exogenous SA. The other (AtICS2) appears to be expressed constitutively, predominantly in the plant vasculature. Here, we characterise the enzymatic activity of both isozymes expressed as hexahistidine fusion proteins inEscherichia coli.We show for the first time that recombinant AtICS2 is enzymatically active. Both isozymes are Mg2+-dependent with similar temperature optima (ca. 33°C) and similarKmvalues for chorismate of 34.3 ± 3.7 and 28.8 ± 6.9 µM for ICS1 and ICS2, respectively, but reaction rates were greater for ICS1 than for ICS2, with respective values forVmaxof 63.5 ± 2.4 and 28.3 ± 2.0 nM s-1and forkcatof 38.1 ± 1.5 and 17.0 ± 1.2 min-1However, neither enzyme displayed isochorismate pyruvate lyase (IPL) activity, which would enable these proteins to act as bifunctional SA synthases, i.e. to convert chorismate into SA. These results show that althoughArabidopsishas two functional ICS enzymes, it must possess one or more IPL enzymes to complete biosynthesis of SA starting from chorismate.
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
authors.forename: Keith M
authors.initials: KM
authors.lastname: Macaulay
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
authors.forename: Geraldine A
authors.initials: GA
authors.lastname: Heath
authors.affiliation: University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
authors.forename: Alessio
authors.initials: A
authors.lastname: Ciulli
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
authors.forename: Alex M
authors.initials: AM
authors.lastname: Murphy
authors.affiliation: University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
authors.forename: John P
authors.initials: JP
authors.lastname: Carr
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K. as25@cam.ac.uk.
authors.forename: Alison G
authors.initials: AG
authors.lastname: Smith
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002827:Chorismic Acid / Q000378:metabolism*; D019295:Computational Biology; D030562:Databases, Protein; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007536:Isomerism; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D029681:Arabidopsis Proteins; C472017:GUN4 protein, Arabidopsis; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C052985:isochorismic acid; D019751:Intramolecular Transferases; C058486:isochorismate synthase; D002827:Chorismic Acid; D008274:Magnesium
keywords: chloroplasts; plant hormones; salicylic acid