title: Transition of phosphopantetheine adenylyltransferase from catalytic to allosteric state is characterized by ternary complex formation in Pseudomonas aeruginosa.
abstract: BACKGROUND
  Phosphopantetheine adenylyltransferase (PPAT) is a rate limiting enzyme which catalyzes the conversion of ATP and pantetheine to dephosphocoenzyme and pyrophosphate. The enzyme is allosteric in nature and regulated by Coenzyme A (CoA) through feedback inhibition. So far, several structures have been solved to decipher the catalytic mechanism of this enzyme.
  
  
  METHODS
  To address catalytic and inhibitory mechanisms of PPAT, structural insights from single crystal X-ray diffraction method were primarily used, followed by biophysical and biochemical analysis.
  
  
  RESULTS
  We have solved the structures of PPAT from Pseudomonas aeruginosa with its substrate analogue AMP-PNP and inhibitor CoA. For the first time, a co-crystal structure of PPAT with Acetyl-CoA (AcCoA) was determined. Enzymatic analysis was performed to decipher the catalytic, allosteric and inhibitory mechanisms involved in regulation of PPAT. Binding affinities of PPAT with its substrates and inhibitors were determined by SPR.
  
  
  CONCLUSIONS
  Previous studies from Escherichia coli and Arabidopsis indicated the inhibitory activity of AcCoA. PPAT-AcCoA structure along with some biochemical methods established AcCoA as an inhibitor to PPAT and illustrated its inhibitory mechanism. Transition from catalytic to allosteric state involves formation of ternary complex. We have studied the structural features of the ternary complex of PPAT along with its product pyrophosphate and inhibitor CoA and validated it with other biophysical and biochemical methods. Extensive analysis of all these 3D structures indicates that changes in side chains R90 and D94 are responsible for transition between catalytic and allosteric inhibitory states.
  
  
  CONCLUSIONS
  These enzymatic studies provide new insights into the allosteric mechanism of PPAT.
authors.affiliation: Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Electronic address: rakesh.2665675@gmail.com.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Chatterjee
authors.affiliation: Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Electronic address: abhisek.mndl@gmail.com.
authors.forename: Abhisek
authors.initials: A
authors.lastname: Mondal
authors.affiliation: Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Electronic address: abasu4@rediffmail.com.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Basu
authors.affiliation: Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Electronic address: saumen_datta@iicb.res.in.
authors.forename: Saumen
authors.initials: S
authors.lastname: Datta
mesh_terms: D000495:Allosteric Site; D055162:Biocatalysis; D008958:Models, Molecular; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
chemical_list: D009713:Nucleotidyltransferases; C022315:pantetheine-phosphate adenylyltransferase
keywords: Enzymatic analysis; Fluorescence; Pantothenate pathway; Protein crystal and X-ray structures; Surface Plasmon Resonance; Ternary complex