Abstract
Phosphopantetheine adenylyltransferase (EC. 2.7.7.3, PPAT) catalyzes the penultimate step of the multistep reaction in the coenzyme A (CoA) biosynthesis pathway. In this step, an adenylyl group from adenosine triphosphate (ATP) is transferred to 4′-phosphopantetheine (PNS) yielding 3′-dephospho-coenzyme A (dpCoA) and pyrophosphate (PPi). PPAT from strain C3 of Klebsiella pneumoniae (KpPPAT) was cloned, expressed and purified. It was crystallized using 0.1 M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82 Å and c = 200.37 Å. The structure was determined using the molecular replacement method and refined to values of 0.208 and 0.255 for Rcryst and Rfree factors, respectively. The structure determination showed the presence of three crystallographically independent molecules A, B and C in the asymmetric unit. The molecules A and B are observed in the form of a dimer in the asymmetric unit while molecule C belongs to the second dimer whose partner is related by crystallographic twofold symmetry. The polypeptide chain of KpPPAT folds into a β/α structure. The conformations of the side chains of several residues in the substrate binding site in KpPPAT are significantly different from those reported in other PPATs. As a result, the modes of binding of substrates, phosphopantetheine (PNS) and adenosine triphosphate (ATP) differ considerably. The binding studies using fluorescence spectroscopy indicated a KD value of 3.45 × 10−4 M for ATP which is significantly lower than the corresponding values reported for PPAT from other species.
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Data availability
The coordinates and structure factors for KpPPAT have been deposited in the Protein Data Bank, under accession numbers 8I8I. All data other than X-ray crystallography supporting the findings of this study are available within the paper and its Supplementary Information.
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Acknowledgements
Authors gratefully acknowledge the support from the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, New Delhi for sponsoring the Synchrotron beamlines at the European Synchrotron Radiation Facility (ESRF), Grenoble, France and the Indian Council of Medical Research (ICMR), New Delhi for a grant to SS. TPS thanks the Science and Engineering Research Board (SERB), New Delhi for the grant of a Distinguished Fellow position to him under the National Science Chair Program of SERB. NA thanks the University Grants Commission, New Delhi for the award of a fellowship to him.
Funding
This work was supported by a project (Grant No. I-1251) from the Indian Council of Medical Research (ICMR), New Delhi to SS and a grant of a Distinguished Fellow position under the National Science Chair (Grant No. SB/DF/002/2019) to TPS from the Science and Engineering Research Board (SERB), New Delhi of the Government of India.
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Conceptualization: SS, NA, TPS; Methodology: NA, PS, SS, TPS; Formal analysis and investigation: NA, SS, TPS; Writing-original draft preparation: TPS, SS, NA; Writing-review and editing: NA, SS, TPS; Funding acquisition: SS, TPS; Resources: SS, TPS, NA, PS; Supervision: SS, TPS.
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Ahmad, N., Sharma, P., Sharma, S. et al. Structure of a novel form of phosphopantetheine adenylyltransferase from Klebsiella pneumoniae at 2.59 Å resolution. Eur Biophys J 53, 147–157 (2024). https://doi.org/10.1007/s00249-024-01703-1
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DOI: https://doi.org/10.1007/s00249-024-01703-1