Mechanism-Based Protein Cross-Linking Probes To Investigate Carrier Protein-Mediated Biosynthesis
2006; American Chemical Society; Volume: 1; Issue: 11 Linguagem: Inglês
10.1021/cb6003965
ISSN1554-8937
AutoresAndrew S. Worthington, Heriberto Rivera, Justin W. Torpey, Matthew D. Alexander, Michael D. Burkart,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoFatty acid, polyketide, and nonribosomal peptide biosynthetic enzymes perform structural modifications upon small molecules that remain tethered to a carrier protein. This manuscript details the design and analysis of cross-linking substrates that are selective for acyl carrier proteins and their cognate condensing enzymes. These inactivators are engineered through a covalent linkage to fatty acid acyl carrier protein via post-translational modification to contain a reactive probe that traps the active site cysteine residue of ketosynthase domains. These proteomic tools are applied to Escherichia coli fatty acid synthase enzymes, where KASI and KASII selectively cross-link ACP-bound epoxide and chloroacrylate moieties. These mechanism-based, protein–protein fusion reagents also demonstrated cross-linking of KASI to type II polyketide ACPs, while nonribosomal peptide carrier proteins showed no reactivity. Similar investigations into protein–protein interactions, proximity effects, and substrate specificities will be required to complete the mechanistic understanding of these pathways.
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