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

Tang, T. M. Simon and Luk, Louis Y. P. ORCID: https://orcid.org/0000-0002-7864-6261 2024. Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization. Faraday Discussions 10.1039/D4FD00002A

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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 feasibility of the AaLS encapsulation approach and encourages further optimization in the design of peptide-ligating artificial organelle in E. coli, aiming for a more effective and stable system for protein modifications.

Item Type:	Article
Date Type:	Published Online
Status:	In Press
Schools:	Chemistry
Publisher:	Royal Society of Chemistry
ISSN:	1359-6640
Funders:	BBSRC (BB/T015799/1), Leverhulme Trust (RPG-2017-195), Royal Society (RG170187) and Wellcome Trust (202056/Z/16/Z)
Date of First Compliant Deposit:	8 April 2024
Date of Acceptance:	16 February 2024
Last Modified:	17 Jun 2024 10:58
URI:	https://orca.cardiff.ac.uk/id/eprint/167809

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