Abstract
Engineering microbial biosynthetic pathways represents a compelling route to gain access to expanded chemical diversity. Carrier proteins (CPs) play a central role in biosynthesis, but the fast motions of CPs make their conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we present a low-resource method to directly reveal carrier protein-substrate interactions. Chemoenzymatic loading of commercially available, alkyne-containing substrates onto CPs enables rapid visualization of the molecular cargo’s local environment using Raman spectroscopy. This method could clarify the foundations of the chain sequestration mechanism, facilitate the rapid characterization of CPs, and enable visualization of the vectoral processing of natural products both in vitro and in vivo.
Highlights
Engineering microbial biosynthetic pathways represents a compelling route to gain access to expanded chemical diversity
For proof-ofconcept experiments, we collected data from three acyl carrier proteins (ACPs) for which chain sequestration information was previously reported via nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics (MD) simulations: the E. coli type II fatty acid synthase (FAS; EcACP), Streptomyces coelicolor type II actinorhodin polyketide synthase (PKS, Act ACP), and the mammalian rat type I FAS (Rat ACP)
For EcACP, MD simulations suggested that an octanoyl acyl chain is the ideal length for complete sequestration of the molecular cargo inside the ACP hydrophobic core[13]
Summary
Engineering microbial biosynthetic pathways represents a compelling route to gain access to expanded chemical diversity. CPs are dynamic proteins that play the most central role during the biosynthesis of pharmaceutically important classes of molecules, such as fatty acids, polyketides, and non-ribosomal peptides (Supplementary Fig. 1) These small proteins interact with virtually all other proteins within the synthase, and they tether a variety of molecular building blocks and intermediates during natural product biosynthesis[2]. The Ppant arm covalently tethers all building blocks and intermediates as thioesters, and its flexibility enables the CP to sequester specific molecular cargoes within its hydrophobic cavity[7] This chain sequestration is believed to protect the growing metabolite from undesired chemical reactions with cytoplasmic components and/or drive other overall conformational changes that can enhance the specificity of a CP for a particular enzymatic partner[8,9]. The alkyne C≡C stretching band is a strong, narrow, and unique signal in the transparent region of the Raman spectrum (close to 2100 cm−1) that does not overlap with other solvent or biomolecular signals from the untagged CP or other proteins
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