Probing the Compatibility of Type II Ketosynthase–Carrier Protein Partners

Abstract

Drug discovery often begins with the screening of large compound libraries to identify lead compounds. Recently, the enzymes involved in the biosynthesis of natural products have been investigated for their potential to generate new, diverse compound libraries. There have been several approaches toward this end, including altering the substrate specificities of the enzymes involved in natural product biosynthesis and engineering functional communication between enzymes from different biosynthetic pathways. While there exist assays to assess substrate specificity of enzymes involved in these pathways, there is no simple method for determining whether enzymes from different synthases will function cooperatively to generate the desired product(s). Herein we report a method which provides insight into both substrate specificity and compatibility of protein-protein interactions between the acyl carrier protein (ACP) and ketosynthase (KS) domains involved in fatty acid and polyketide biosynthesis. Our technique uses a one-pot chemoenzymatic method to generate post-translationally modified ACPs capable of covalently interacting with KS domains from different biosynthetic systems. The extent of interaction between ACPs and KSs from different systems is easily detected and quantified by a gel-based method. Our results are consistent with previous studies of substrate specificity and ACP-KS binding interactions and provide new insight into unnatural substrate and protein interactions.

Chapter 17 Bacterial Fatty Acid Synthesis and its Relationships with Polyketide Synthetic Pathways

“…Both versions of E. coli AcpP also reacted with S. maritimus EncAB. However, only a partially reciprocal result was seen in that FabF failed to react with either version of EncC whereas FabB reacted only with EncC carrying the shorter analogue (Worthington et al, 2008). These findings raise the question of whether or not EncC functions in both the FAS and entericin pathways of S. maritimus even though the ACP is encoded in the gene cluster of the latter pathway.…”

“…Recently a new approach to test the compatibility of the ACPs of type II FAS and PKS systems has been developed (Worthington et al, 2008). This approach uses modified ACP species that carry a reactive electrophile in place of the thiol of the 4’-PP moiety.…”

Harnessing natural product assembly lines: structure, promiscuity, and engineering

“…Mechanism-based crosslinkers have proven popular tools to probe various intramodular interactions between the ACP/PCP and other components including domains from NRPSs, fatty acid synthases (FASs) and fungal non-reducing PKSs [58,87]. For example, unnatural phosphopantetheine analogues modified with chemical handles for crosslinking have been successfully used to investigate ACP protein-protein interactions to better understand the protein interactions involved with chain elongation ( Figure 3a ) [141,143]. The efficiency of mechanism-based crosslinking to the KS usually correlates with the strength of the ACP:KS interaction [142].…”

Utilizing Mechanistic Cross-Linking Technology To Study Protein–Protein Interactions: An Experiment Designed for an Undergraduate Biochemistry Lab

“…1,4 These crosslinking probes covalently trap the ACP with catalytically-functional partner proteins to form a stable complex (Figure 1 and S1). 5 …”