Probing the compatibility of type II ketosynthase-carrier protein partners.
Drug discovery often begins with the screening of large compound libraries to identify lead compounds. Recently, the enzymes that are 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 the 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 that 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 that are 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.
Worthington
Andrew S
AS
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.
Hur
Gene H
GH
Meier
Jordan L
JL
Cheng
Qian
Q
Moore
Bradley S
BS
Burkart
Michael D
MD
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D029785:Cyperaceae / Q000201:enzymology*; D015195:Drug Design; D004926:Escherichia coli / Q000201:enzymology*; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010204:Pantetheine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D048630:Polyketide Synthases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D010204:Pantetheine; D048630:Polyketide Synthases; D054889:Fatty Acid Synthase, Type II; C003129:4'-phosphopantetheine