9QVS

Crystal structure of the CtaG_D144N variant from Ruminiclostridium cellulolyticum (P2(1)-small)

Summary for 9QVS

• Entry DOI: 10.2210/pdb9qvs/pdb
• Related: 9QUZ
• Descriptor: Butirosin biosynthesis protein H N-terminal domain-containing protein, SODIUM ION (3 entities in total)
• Functional Keywords: amide bond formation; antibiotics; biosynthesis; carrier proteins; enzymes, nonribosomal peptide synthetases, ligase
• Biological source: Ruminiclostridium cellulolyticum
• Total number of polymer chains: 1
• Total formula weight: 37213.83

Authors

Gude, F.,Bohne, A.,Dell, M.,Franke, J.,Dunbar, K.L.,Groll, M.,Hertweck, C. (deposition date: 2025-04-11, release date: 2025-10-01, Last modification date: 2026-02-18)

Primary citation

Gude, F.,Bohne, A.,Dell, M.,Franke, J.,Dunbar, K.L.,Groll, M.,Hertweck, C.
Distal peptide elongation by a protease-like ligase and two distinct carrier proteins.
Chem, 12:None-None, 2026

PubMed Abstract: Closthioamide (CTA) is a potent antibiotic with a unique polythioamide scaffold produced by . Unlike classical non-ribosomal peptide synthetases (NRPSs), which use modular adenylation and condensation domains, CTA biosynthesis proceeds through non-canonical standalone enzymes. Central to this process is the papain-like ligase CtaG, which catalyzes amide bond formation between two distinct peptidyl carrier proteins (PCPs): CtaH, presenting para-hydroxybenzoic acid (PHBA), and CtaE, carrying a tri-β-alanine ((βAla)) chain. Using biochemical assays, chemical probes, crystallography, and mutational analysis, we show that CtaG operates via a ping-pong mechanism involving an enzyme-bound intermediate. A single substrate tunnel mediates directional transfer, enabling distal chain elongation that mirrors solid-phase peptide synthesis. Structure-based genome mining revealed homologous enzymes in the biosynthetic pathways of petrobactin, butirosin, and methylolanthanin. Together, our findings uncover a previously overlooked class of thiotemplated ligases and provide a mechanistic blueprint for engineering ribosome-independent peptide assembly lines.

PubMed: 41641319
DOI: 10.1016/j.chempr.2025.102740

Experimental method

X-RAY DIFFRACTION (1.45 Å)