8JPT

Crystal Structure of the acyltransferase domain from the eighth module of the spinosad polyketide synthase

Summary for 8JPT

• Entry DOI: 10.2210/pdb8jpt/pdb
• Descriptor: Polyene macrolide polyketide synthase (2 entities in total)
• Functional Keywords: acyltransferase domain, transferase
• Biological source: Saccharopolyspora spinosa
• Total number of polymer chains: 3
• Total formula weight: 132344.02

Authors

Huang, S.,Zheng, J. (deposition date: 2023-06-12, release date: 2024-06-19, Last modification date: 2025-07-02)

Primary citation

Huang, S.,Ji, H.,Zheng, J.
Structural and computational insights into the substrate specificity of acyltransferase domains from modular polyketide synthases.
Febs J., 291:3839-3855, 2024

PubMed Abstract: Polyketides are natural products synthesized by polyketide synthases (PKSs), where acyltransferase (AT) domains play a crucial role in selection of extender units. Engineering of AT domains enables the site-specific incorporation of non-natural extender units, leading to generation of novel derivatives. Here, we determined the crystal structures of AT domains from the fifth module of tylosin PKS (TylAT5) derived from Streptomyces fradiae and the eighth module of spinosad PKS (SpnAT8) derived from Saccharopolyspora spinosa, and combined them with molecular dynamics simulations and enzyme kinetic studies to elucidate the molecular basis of substrate selection. The ethylmalonyl-CoA-specific conserved motif TAGH of TylAT5 and the MMCoA-specific conserved motif YASH of SpnAT8 were identified within the substrate-binding pocket, and several key residues close to the substrate acyl moiety were located. Through site-directed mutagenesis of four residues near the active site, we successfully reprogrammed the specificity of these two AT domains toward malonyl-CoA. Mutations in TylAT5 enhanced its catalytic activity 2.6-fold toward malonyl-CoA, and mutations in SpnAT8 eliminated the substrate promiscuity. These results extend our understanding of AT substrate specificity and would benefit the engineering of PKSs.

PubMed: 38922792
DOI: 10.1111/febs.17206

Experimental method

X-RAY DIFFRACTION (3.26 Å)