9KPY

Structure of Phosphopantetheine adenylyltransferase (PPAT) from Enterobacter spp. with the expression tag bound in the substrate binding site of a neighbouring molecule at 2.20 A resolution.

Replaces:  8I8J

Summary for 9KPY

• Entry DOI: 10.2210/pdb9kpy/pdb
• Descriptor: Phosphopantetheine adenylyltransferase, Heptapeptide, PHOSPHONOACETIC ACID, ... (5 entities in total)
• Functional Keywords: coad, ppat, transferase, coenzyme a biosynthesis, expression tag
• Biological source: Enterobacter sp. 638; More
• Total number of polymer chains: 7
• Total formula weight: 117239.79

Authors

Ahmad, N.,Sharma, P.,Sharma, S.,Singh, T.P. (deposition date: 2024-11-24, release date: 2024-12-11, Last modification date: 2025-08-20)

Primary citation

Ahmad, N.,Kumar, V.,Goel, V.K.,Sharma, P.,Sharma, S.,Singh, T.P.
Protein-tags and their fragments as potent inhibitors of enzymes: Structure of the ternary complex of phosphopantetheine adenylyltransferase from Enterobacter spp. with tag-peptides and phosphonoacetic acid at 2.20 angstrom resolution.
Protein Sci., 34:e70216-e70216, 2025

PubMed Abstract: Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step of coenzyme A (CoA) biosynthesis pathway by transferring the adenylyl group from adenosine triphosphate (ATP) to 4'-phosphopantetheine (PNS), yielding 3'-dephosphocoenzyme A and pyrophosphate. In this study, the recombinant PPAT from Enterobacter spp. strain 638 (EbPPAT) was purified and co-crystallized with phosphonoacetic acid (PAE). The structure showed the presence of three homodimers AB, CD, and EF in the asymmetric unit. The 14 extra N-terminal residues (Met-14 to Ser-1, 14-mer peptide) from the expression tag were observed in Molecules B and F. These tag-peptides occupied the PNS-binding sites of adjacent Molecules A and E, respectively. Additionally, a heptapeptide (Met-14 to Gly-8) was also observed in the PNS-binding site of Molecule C. Furthermore, two PAE molecules were present in the ATP-binding sites of Molecules B, D, and F, whereas a single PAE molecule was found in Molecules A, C, and E. This showed that tag-peptides blocked the PNS-binding site while PAE blocked the ATP-binding sites. Three peptides of the tag, including 14-mer (Met-14 to Ser-1), heptapeptide (Met-14 to Gly-8) and pentapeptide (Met-14 to Thr-10) were synthesized, and their binding affinities were estimated, which showed the K values of 5.5 × 10, 1.8 × 10, and 7.3 × 10 M, respectively. PAE molecules bound to EbPPAT in the ATP-binding sites with a K of 4.77 × 10 M. This is the first structure of PPAT with peptides bound in the substrate-binding sites, indicating a novel approach to design peptide inhibitors.

PubMed: 40689715
DOI: 10.1002/pro.70216

Experimental method

X-RAY DIFFRACTION (2.2 Å)