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.

Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking

“…The difficulty of probing and interrogating the weak and transient interactions between ACP's and KS's is exemplified by the ongoing elegant work of Burkart and co-workers, which relies on the design and synthesis of specific mechanism-based probes that covalently cross-link the ACP and cognate interaction partner. 4,5,29,30 Clearly, additional tools need to be developed and evaluated for studying protein interactions among FAS's and PKS's, particularly for those biosynthetic systems with unusual substrate specificities, whereby effective cross-linkers might be difficult to discover. Macromolecular interactions of proteins in other biological systems have often been probed via the introduction of photocross-linking unnatural amino acids.…”

Catalytic Turnover-Based Phage Selection for Engineering the Substrate Specificity of Sfp Phosphopantetheinyl Transferase

“…[55][56][57] These CoA analogues were synthesized in a chemoenzymatic procedure with the use of pantetheine derivatives that were sequentially modified by the three enzymes involved in CoA biosynthetic pathway-pantothenate kinase (PanK), phosphopantetheine adenyltransferase (PPAT), and dephosphocoenzyme A kinase (DPCK). 58,59 PanK phosphorylates pantetheine derivatives and PPAT couples phosphopantetheine to the α-phosphate of ATP to afford dpCoA analogues with Ppant substitution.…”

Malonyl carba(dethia)‐ and Malonyl oxa(dethia)‐coenzyme A as Tools for Trapping Polyketide Intermediates

“…The methodology for polyketide-chain termination also prevents the A C H T U N G T R E N N U N G intramolecular formation of pyrone products, [48] and it can be further extended to other PKS. Analogues of pantetheines have recently been used to tag carrier proteins to trap transient protein-protein interactions [49] or to manipulate the carrier geometry. [50] We anticipate that upon loading nonhydrolyzable malonyl carbaA C H T U N G T R E N N U N G (dethia)pantetheine (from 2) onto the acyl carrier protein (ACP) using phosphopantetheinyl transferase Sfp, [51] our approach will prove to be a powerful tool for studying such complex PKS systems.…”