Isochorismate pyruvate lyase
Isochorismate Pyruvate LyaseIsochorismate Pyruvate Lyase
Gateway for Substrate Entry and Product Exit
Significance of the Isochorismate Isochorismate is the end product of the shikimate pathway that is essential for the synthesis of many primary and secondary metabolites. It is synthesized from chorismate by isochorismate synthase enzyme. Chorismate is the common precursor for synthesis of aromatic amino acids, cofactors, phenazines and siderophores. Many enzymes are involved in the pathway and these enzymes are present in microbes and plants and absent in mammals and provide potential targets for antimicrobial drugs and herbicides.Isochorismate pyruvate Lyase (IPL) is one such enzyme.
FunctionFunction
It is involved in the catalysis of the transformation of isochorismate to pyruvate and salicylate. This reaction is the committed step in the biosynthesis of salicylate-based siderophores in several pathogenic bacteria. In plants, salicylate produced from isochorismate is important for the plant defense mechanisms known as local and systemic acquired resistance It has also been found to have secondary activity of catalyzing the transformation of chorismate to prephenate (chorismate mutase activity), although this is weak. The physiological role of IPL or PchB uses a pericyclic hydrogen transfer mechanism to produce salicylate from isochorismate as opposed to the notion that general base or acid catalysis would occur. This property resembles chorismate mutase enzyme that catalyze pericyclic reactions. IPL is a structural homolog of chorismate mutase despite very low sequence identity (approx 20%). The similarity is restricted to the active sites which are conserved (Annu. Rev. Biophys. 2008. 37:153–73) and it has been found that chorismate mutase cannot be mutated to acquire IPL activity. IPL has been considered for improvement of its secondary activity computationally using hybrid quantum mechanics/ Molecular mechanics (J. AM. CHEM. SOC. 2008, 130, 2894-2895).Homologous genes are found in other microorganisms. It is also involved in bacterial siderophore synthesis. Peter Kast et al, propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs.
StructureStructure
This is a 101-residue chain made of three alpha helices. Some of the regions overlap with the regions of RdgC base domain which is a DNA binding recombination association protein.The quaternary structure of PchB was found to be dimeric as it is for EcCM (the homologous Chorismate Mutase), and most catalytic residues in the active site of EcCM are conserved in PchB. Moreover, it was shown that pchB complements the CM deficiency of an E. coli mutant strain and that PchB has low CM activity in vitro.
ChemistryChemistry
The transformation of isochorismate to prephenate is a pericyclic reaction. IPL catalyzes the elimination of the enolpyruvyl side chain from isochorismate to give salicylate and pyruvate. This type of aromatization reaction is generally formulated as a baseinitiated process, but a dissociative mechanism involving initial cleavage of the C-O bond to give an ion pair intermediate is conceivable. A concerted pericyclic pathway, in which the hydrogen atom at C2 is transferred to C9 of the side chain simultaneous with C-O cleavage, which represents a third possibility, has been proposed as well. The reaction is a one step mechanism (J. AM. CHEM. SOC. 2005, 127, 15002-15003).
Correlation of structure with functionCorrelation of structure with function
ApplicationsApplications
Bacterial genes can be introduced into plants to increase the accumulation of the desired compounds. Salicylic acid content in Arabidopsis thaliana can be manipulated by expressing an engineered bacterial salicylate synthase by the fusion of two bacterial genes pchA and pchB from the human pathogen, Pseudomonas aeruginosa, that encode isochorismate synthase and isochorismate pyruvate lyase. These are expressed under a constitutive promoter.
CrystallizationCrystallization
X-ray crystallization is performed by hanging drop method for the apo, open and closed structures. The apo structure has nitrate in it in the regions that have the pyruvate in the bound structures. In the case of the bound structures, immediately prior to crystallization dilution in pyruvate is performed. These proteins carry the 6His Tag, but on crystallization, these are proteolyzed in the drop. Molecular replacement of the Apo with the structure of ECM (chorismate mutase) was done. Crystals of the bound structure are obtained by growing in PEG 3350. The resolution abtained for the Apo structure is 2.35 Angstroms and for the pyruvate bound structure it is 1.95 Angstroms. The residues in the loop that connects the first helix to the second has no residues lighting up in the data, depicting the disorderliness. These residues are present in case of the pyruvate bound structures in which case they are ordered.
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Click to see sidechains in the morph too.
To the right we can see the structure from apo to open pyruvate bound form. The representation from open to closed is given too.
Isochorismate-Pyruvate Lyase: open conformation with pyruvate bound morphing to closed conformation with pyruvate bound
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Active SiteActive Site
The difference between the apo and the ligand bound open and close structures is in the ligand binding and the loop connecting the first and the second helix. In case of the open form, the loop is disordered and in case of the closed form it is ordered. The apo structure has nitrate ion in the active site (that is occupied by pyruvate) due to ammonium nitrate reservoir buffer during crystallization.
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