Mechanism-based protein cross-linking probes to investigate carrier protein-mediated biosynthesis.
Fatty 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.
Worthington
Andrew S
AS
Rivera
Heriberto
H
Torpey
Justin W
JW
Alexander
Matthew D
MD
Burkart
Michael D
MD
D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry; D014176:Protein Biosynthesis / Q000502:physiology*; D017433:Protein Structure, Secondary / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins