title: Mechanism-based protein cross-linking probes to investigate carrier protein-mediated biosynthesis.
abstract: 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.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Worthington
authors.forename: Heriberto
authors.initials: H
authors.lastname: Rivera
authors.forename: Justin W
authors.initials: JW
authors.lastname: Torpey
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Alexander
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
mesh_terms: 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
chemical_list: D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins