Volume 252, 2024

Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization

Abstract

Peptide asparaginyl ligases (PALs) hold significant potential in protein bioconjugation due to their excellent kinetic properties and broad substrate compatibility. However, realizing their full potential in biocatalytic applications requires precise control of their activity. Inspired by nature, we aimed to compartmentalize a representative PAL, OaAEP1-C247A, within protein containers to create artificial organelles with substrate sorting capability. Two encapsulation approaches were explored using engineered lumazine synthases (AaLS). The initial strategy involved tagging the PAL with a super-positively charged GFP(+36) for encapsulation into the super-negatively charged AaLS-13 variant, but it resulted in undesired truncation of the enzyme. The second approach involved genetic fusion of the OaAEP1-C247A with a circularly permutated AaLS variant (cpAaLS) and its co-production with AaLS-13, which successfully enabled compartmentalization of the PAL within a patch-work protein cage. Although the caged PAL retained its activity, it was significantly reduced compared to the free enzyme (∼30–40-fold), likely caused by issues related to OaAEP1-C247A stability and folding. Nevertheless, these findings demonstrated the feasibility of the AaLS encapsulation approach and encourage further optimization in the design of peptide-ligating artificial organelles in E. coli, aiming for a more effective and stable system for protein modifications.

Graphical abstract: Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization

  • This article is part of the themed collection: Biocatalysis

Associated articles

Article information

Article type
Paper
Submitted
08 jan 2024
Accepted
16 feb 2024
First published
20 feb 2024
This article is Open Access
Creative Commons BY license

Faraday Discuss., 2024,252, 403-421

Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization

T. M. S. Tang and L. Y. P. Luk, Faraday Discuss., 2024, 252, 403 DOI: 10.1039/D4FD00002A

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