3X1K: Crystal Structure Of Phosphoapantetheine Adenylyltransferase Ppat/coad With Amp-pnp From Pseudomonas Aerugonosa

Citation:
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. CONCLUSION: 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. GENERAL SIGNIFICANCE: These enzymatic studies provide new insights into the allosteric mechanism of PPAT.
PDB ID: 3X1KDownload
MMDB ID: 134345
PDB Deposition Date: 2014/11/20
Updated in MMDB: 2015/11
Experimental Method:
x-ray diffraction
Resolution: 2.55  Å
Source Organism:
Similar Structures:
Biological Unit for 3X1K: trimeric; determined by author and by software (PISA)
Molecular Components in 3X1K
Label Count Molecule
Proteins (3 molecules)
3
Phosphopantetheine Adenylyltransferase
Molecule annotation
Chemicals (16 molecules)
1
3
2
10
3
2
4
1
* Click molecule labels to explore molecular sequence information.

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